Molecular mechanism of naturally-encoded signaling-bias at the complement anaphylatoxin receptors

GPCRs, ACRs, signaling-bias, β-Arrestins, anaphylatoxin receptors, complement 22 cascade, drug discovery 23 24 25 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 2

26 The conceptual framework of biased signaling has revolutionized our understanding of GPCR 27 signaling and regulatory paradigms, and greatly impacted the efforts focused on the discovery 28 of GPCR-targeted therapeutics. However, the mechanistic basis of biased signaling remains 29 primarily defined based on synthetic ligands and receptor mutants with relatively limited 30 progress in understanding naturally-encoded signaling-bias. Here, we present fundamental 31 molecular and structural insights into naturally -encoded signaling -bias at the complement 32 anaphylatoxin C5a receptors namely, C5aR1 and C5aR2. We first discover that C5a-d-Arg, the 33 naturally-occurring version of C5a lacking the terminal arginine, exhibits robust G -protein 34 signaling-bias at C5aR1 , characterised by attenuated βarr recruitment . This signaling -bias 35 manifests in both cytokine release from primary human immune cells, and in vivo, during 36 neutrophil mobilization . We combine the cryo -EM structures of C5a/C5a -d-Arg-C5aR1 37 complexes with MD simulation, site -directed mutagenesis, and cellular experiments to 38 elucidate that the G-protein-bias exhibited by C5a-d-Arg results from a distinct orientation of TM7 39 and helix 8 in C5aR1 leading to inefficient GRK recruitment and receptor phosphorylation. 40 Next, we determine the first cryo -EM structures of C5aR2, a naturally -encoded β-arrestin-41 biased receptor, in an apo state, complexed with the natural agonist s C5a and C5a-d-Arg, and 42 three peptide agonists including a first -in-class, newly discovered C5aR2 -selective agonist, 43 R8Y. These structural snapshots reveal key differences between the binding of C5a and C5a-44 d-Arg to C5aR1 and C5aR2, and provide a molecular basis of functional specialization at these 45 two receptors. Moreover, the structural insights also allow us to decipher the molecular basis 46 of naturally-encoded signaling-bias at C5aR2 originating from a shallower cytoplasmic 47 interface with hydrophobic interior pocket that is not permissive to efficient G-protein-coupling 48 and activation. Finally, we also engineer and characterize loss-of-function and gain-of-function 49 variants of C5aR1 and C5aR2, which in turn corroborate and validate the structural 50 observations presented here. Collectively, our findings offer crucial insights into previously 51 lacking molecular mechanisms of the naturally-encoded signaling-bias at GPCRs, which have 52 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 3 broad implications not only for the general framework of biased -signaling, but also for novel 53 therapeutic design. 54

55 G protein-coupled receptors (GPCRs) constitute a large superfamily of seven transmembrane 56 receptors (7TMRs) with a direct involvement in a broad array of physiological and 57 pathophysiological processes, which also makes them highly sought -after drug targets 1,2. 58 Upon agonist -stimulation, GPCRs typically couple to , and signal through , heterotrimeric 59 G-proteins and β-arrestin (βarrs), and it has been possible to design synthetic ligands capable 60 of preferentially activating one of these transducers 3,4. These ligands are known as biased -61 agonists and the phenomenon of preferential activation of a specific transducer as biased 62 agonism, and their therapeutic promise has brought about a paradigm change in GPCR -63 targeted novel drug discovery 5-7. The conceptual framework of biased -signaling is based 64 primarily on synthetic ligands and receptor mutants for the most commonly studied systems, 65 and the examples of naturally-encoded biased ligands are rather limited4,5,7-10. This represents 66 a key knowledge gap and poses an important caveat in fully appreciating the physiological 67 implications of this therapeutically important paradigm. Interestingly, the complement 68 anaphylatoxins and their cognate receptors encoded in the complement cascade constitute 69 an intriguing system to probe the naturally -encoded ligand-induced and receptor -mediated 70 signaling-bias11-14. 71 The complement cascade plays a critical role in the innate immune response 72 mechanisms, especially in the complex landscape of host -pathogen interaction s, as a 73 protective mechanism 15,16. The potent anaphylatoxins referred to as C3a and C5a are 74 generated in the final steps by the proteolytic cleavage of complement proteins C3 and C5, 75 and they activate three different 7TMRs known as C3aR, C5aR1 and C5aR2 to exert the 76 functional outcomes such as chemotaxis, degranulation, and cytokine production17-19. These 77 receptors are expressed by a variety of immune cells such as macrophages and neutrophils, 78 and upon activation by the corresponding anaphylatoxins, mediate a wide array of functional 79 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 4 responses14,20-24. Their excessive and sustained activation is often associated with multiple 80 pathophysiological conditions including sepsis, autoimmune disorders, rheumatoid arthritis, 81 and multiple sclerosis, making them important drug targets for novel therapeutics 14,23,25-30. 82 Interestingly, the terminal arginine residue in C3a and C5a are cleaved by carboxypeptidases 83 to generate C3a -d-Arg and C5a -d-Arg, respectively, and it is commonly believed to be a 84 mechanism to dampen the inflammatory response as the terminal arginine is critical for the 85 binding of C3a/C5a to the corresponding receptors31-34. However, there are indications in the 86 literature that C5a-d-Arg may still bind to C5aR1 and C5aR2 and exert functional responses 23. 87 Therefore, a systematic and comprehensive exploration of C3a -d-Arg/C5a-d-Arg interaction with 88 their cognate receptors is essential to resolve uncertainties regarding their functional and 89 physiological relevance. Moreover, a molecular understanding of these interactions guide the 90 design of receptor subtype-selective ligands, especially for C5aR2 which currently lacks 91 potent tool compounds, and help segregate overlapping and distinct functions of C5aR1 and 92 C5aR224. 93 There are a set of 7TMRs that lack functional G -protein-coupling despite having an 94 overall architecture similar to GPCRs, and these are classified as Atypical Chemokine 95 Receptors (ACKRs) as they recognize chemokines as their natural agonists35-39. While four of 96 these receptors namely ACKR2-5 couple to βarrs, one of these, ACKR1, also known as the 97 Duffy antigen receptor for chemokines (DARC), lacks a measurable coupling to βarrs as well40. 98 The sub-family of these so called non-canonical GPCRs is further expanded by C5aR2, which 99 also lacks functional G -protein-coupling but maintains robust βarr recruitment despite being 100 activated by C5a and C5a -d-Arg12. Taken together, these five receptors , i.e., ACKR2-5 and 101 C5aR2 constitute a sub-family referred to as Arrestin-Coupled Receptors (ACRs), and present 102 an excellent system to study the intricacies of naturally-encoded signaling-bias at the receptor 103 level. In particular, most of these ACRs share a natural agonist with a prototypical GPCR, 104 thereby presenting a GPCR-ACR pair to directly compare the commonalities and differences 105 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 5 in their ligand binding, conformational changes, and transducer-coupling to elucidate the key 106 principles of biased signaling41. 107 Here, we demonstrate using a combination of cellular, biochemical, and 108 pharmacological approach es that C5a -d-Arg acts as a robust G -protein-biased agonist at 109 C5aR1, and the transducer-coupling-bias is linked to distinct cellular and functional outcomes. 110 We present the cryo -EM structures of C5a -d-Arg-bound C5aR1 and combine the structural 111 insights with biochemical experiments to uncover the molecular mechanism driving the 112 signaling-bias. Moreover, we also determine the first cryo-EM structures of the C5aR2 in apo-113 state, in complex with C5a, and a set of peptide agonists including a first -in-class, C5aR2-114 selective agonist. These structural snapshots elucidate the molecular basis of intrinsic 115 signaling-bias encoded at C5aR2 , and also uncover the design principles that allow us to 116 engineer sub-type selective and signaling-biased C5a variants at C5aR1 and C5aR2. 117

118 C5a-d-Arg is a naturally-encoded biased agonist at C5aR1 119 As reported previously42 and reproduced here ( Figure 1A -C, S1A-H), we observed that 120 contrary to the broadly presented notion in the literature 33,43,44, C5a-d-Arg activates G-proteins 121 with potency and efficacy nearly indistinguishable from C5a while it is substantially attenuated 122 in βarr recruitment. The calculation of bias-factor further corroborates these observations, and 123 establishes C5a-d-Arg as a G-protein-biased agonist at C5aR1. Similar to the data observed in 124 HEK-293T cells, we observed an equivalent Ca 2+ response for both C5a and C5a -d-Arg in 125 primary human monocyte derived macrophages (HMDMs) and mouse bone marrow derived 126 macrophages (BMDMs) ( Figure 1D -E). However, ERK1/2 phosphorylation in HEK -293, 127 HMDMs, and BMDMs, and RhoA activation in HEK-293 cells was significantly attenuated for 128 C5a-d-Arg compared to C5a (Figure 1F-I, S1I-J, S1K-L). Interestingly, C5a and C5a-d-Arg elicited 129 a similar response in terms of IL -8 release from human macrophages (Figure 1J) while the 130 bell-shaped dose-response typically observed for C5a in the PMN (polymorphonuclear 131 leukocytes) migration assay45 was not observed for C5a-d-Arg (Figure 1K). Moreover, plasma-132 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 6 purified C5a-d-Arg recapitulated a similar pattern as that of the recombinant C5a-d-Arg in terms of 133 Ca2+ influx in HMDMs ( Figure 1L), βarr2 recruitment in HEK -293 cells measured by BRET -134 assay (Figure 1M), pERK1/2 phosphorylation in HMDM (Figure 1N) and BMDMs (Figure 135 1O), IL-8 release (Figure 1P), and PMN migration (Figure 1Q). In order to further link these 136 in vitro observations with an in vivo readout, we administered C5a and C5a-d-Arg in mice to 137 induce bone marrow neutrophil mobilization into the blood. We observed a significant 138 reduction in neutrophil blood mobilization for C5a-d-Arg compared to C5a ( Figure 1R), which 139 aligns with a functional -bias encoded by C5a -d-Arg. Interestingly, pre -dosing mice with the 140 C5aR1-selctive antagonist PMX20546, followed by the administration of C5a and C5a-d-Arg, 141 reduced neutrophil blood mobilization as expected (due to blockade of C5aR1), but more 142 importantly, there was no apparent difference between C5a vs. C5a -d-Arg (Figure 1R). These 143 data indicate that the attenuation of βarr recruitment exhibited by C5a -d-Arg translates into a 144 functional response (i.e., neutrophil mobilization) in vivo, and that the residual response after 145 C5aR1 blockade likely arises from the second C5a receptor, C5aR2, which is discussed in a 146 subsequent section. Taken together, these data corroborate an intrinsic functional -bias 147 encoded by C5a-d-Arg at the human and mouse C5aR1, and also establish it as one of the very 148 few naturally-encoded biased agonists identified till date. 149 Molecular mechanism of signaling-bias of C5a-d-Arg 150 In order to understand the molecular basis of signaling -bias exhibited by C5a -d-Arg, we 151 determined the cryo -EM structure of mC5a -mC5aR1-G-protein and mC5a -d-Arg-mC5aR1-G-152 protein complexes (overall snapshot presented here in Figure 1S)42. Similar to the hC5a-d-Arg-153 hC5aR1-G-protein complex reported earlier11, we observed that the last three amino acids in 154 mC5a-d-Arg namely Q71, L72, and G73 slide into a binding pocket that is occupied by the 155 terminal arginine in mC5a to compensate for critical interactions in the pocket ( Figure 1S). 156 Therefore, it is likely that the differences observed at the functional level are encoded by 157 differential dynamics of these interactions. To probe this hypothesis, we employed molecular 158 dynamics (MD) simulations47,48 using the structural templates of C5a and C5a-d-Arg complexes. 159 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 7 We observed that R74 in C5a forms extensive polar interactions in the orthosteric binding site 160 with Arg1754.64, Arg2065.42, and Asp2827.35 while G73 in C5a -d-Arg can only partly recapitulate 161 these interactions, for example, with Arg1754.64 and Arg2065.42, but not with Asp2827.35 (Figure 162 2A and S2A). (To avoid any ambiguity, one-letter amino acid code for ligand and three-letter 163 amino acid code for receptor residues have been used) . Interestingly, the loss of C5a-d-Arg 164 interaction with Asp282 7.35 substantially alters the contact network in the orthosteric binding 165 pocket of the receptor. In particular, we observed that Arg175 4.64 and Arg2065.42 maintained 166 some interaction with C5a-d-Arg during the course of simulation, while Asp2827.35 did not engage 167 with the ligand (Figure 2A). In contrast, all three residues maintained a stable interaction with 168 C5a. Consistent with the MD simulation, site -directed mutagenesis of Arg175 4.64 and 169 Arg2065.42 to alanine resulted in a significant loss of G -protein-coupling for C5a-d-Arg while 170 the effects were rather modest for C5a (Figure 2B, S2B and S2F). Notably, all three mutations 171 dramatically attenuated βarr recruitment for both C5a and C5a-d-Arg (Figure 2C, S2C and S2F). 172 These observations suggest that a loss of either one of these residues can be tolerated for 173 C5a-induced G-protein activation due to sustained interaction with the remaining two residues, 174 while the loss of even one of these interactions has a more drastic effect on C5a-d-Arg-induced 175 G-protein-coupling. Conversely, engaging all three residues is critical for βarr recruitment by 176 either ligand. 177 Structural comparison of C5a-C5aR1 and C5a-d-Arg-C5aR1 revealed an overall similar 178 structure with comparable spatial positioning of the C5a-d-Arg core domains on the receptor with 179 a small linear shift, and the carboxy terminus residues of C5a-d-Arg adopting a similar hook-like 180 conformation as observed in C5a -C5aR1 (Figure S2D-E). Interestingly, helix 8 of C5aR1 in 181 the C5a -d-Arg-bound structure undergoes a rotation of ~120º and a linear shift of ~5 Å (as 182 measured from the Cα of Ser3147.36) towards the cytoplasmic portion of TM1 compared to that 183 in C5a-C5aR1 structure (Figure 2D). Our MD simulation study also suggests that disrupting 184 the contact pattern in the orthosteric binding pocket impacts the conformation space explored 185 by TM7, wherein the loss of ligand interaction with TM7 (Asp2827.35) leads to a significant shift 186 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 8 of TM7 (Figure 2E). These differences between the C5a vs. C5a-d-Arg-bound structures can be 187 quantified in terms of the distance between TM2 and TM7 (Ile962.64 and Phe2757.28), which is 188 markedly larger in the C5a-d-Arg-bound C5aR1 (Figure 2E). In our mC5a/mC5a-d-Arg structures, 189 we also observe Q71 of mC5a establishes hydrogen bonds with Ser 284 (S7.36) of mC5aR1, 190 which is lost in mC5a -d-Arg-bound mC5aR1 ( Figure 2F). Interestingly, HDX -MS experiments 191 also show significant decrease in deuterium exchange in the N-terminus of TM1 and TM7, in 192 mC5a-bound mC5aR1, compared to mC5a -d-Arg-mC5aR1, with the decrease being more 193 profound in TM7. This observation aligns with the structural interpretation that TM7 is 194 stabilized by the interactions established by mC5a with the receptor, and the loss of this 195 interaction results in flexibility in TM7 of mC5a -d-Arg-bound mC5aR1 (Figure 2G and S2G). It 196 is likely that this conformational change in TM7 propagates down to the intracellular side of 197 the receptor and impacts the arrangement of helix 8. 198 A previously reported structure of NTSR1 in complex with GRK2 demonstrates a direct 199 engagement of helix 8 in the receptor with the N-terminus of GRK2, thereby holding GRK2 in 200 a spatial position to facilitate efficient receptor phosphorylation49. Using this as a reference, it 201 is plausible that the spatial rearrangement of helix 8 in C5a -d-Arg-bound C5aR1 may impact 202 efficient GRK engagement with the receptor. Indeed, a direct measurement of C5aR1 203 interaction with GRKs using a NanoBiT assay revealed a significant attenuation of receptor -204 GRK engagement upon stimulation with C5a -d-Arg compared to C5a (Figure 2H). This further 205 translates into inefficient phosphorylation of the C5aR1 as reflected in bulk phosphorylation 206 measured using the pIMAGO assay, and site -specific phosphorylation measured using 207 phospho-site-specific antibodies (Figure 2I). Taken together, these data provide a molecular 208 explanation for attenuated βarr recruitment as observed in the cellular context upon 209 stimulation of the receptor with C5a-d-Arg compared to C5a, and ensuing signaling-bias. 210 Structural basis of C5a-binding to C5aR2 211 Next, we focused our attention on C5aR2 in order to understand the molecular basis of 212 naturally-encoded βarr-bias of this receptor despite binding the same natural agonists as 213 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 9 C5aR1, i.e., C5a and C5a-d-Arg (Figure 3A). Interestingly, we observed that unlike C5aR1, both 214 C5a and C5a-d-Arg display nearly identical potency and efficacy at C5aR2 in the βarr recruitment 215 assays ( Figure 3B and S3A). These observations in transfected cells align well with the 216 in vivo data where the residual neutrophil mobilization upon C5a and C5a-d-Arg stimulation with 217 C5aR1 blockade, likely mediated by C5aR2, are nearly identical ( Figure 1R). The structural 218 analysis of C5aR2 using cryo -EM poses a challenge due to lack of G -protein-coupling, and 219 previous efforts to isolate C5aR2-βarr complexes stabilized by Fab30 yielded only miniscule 220 amounts of ternary complexes suitable for high -resolution analysis 12. While purifying 221 recombinant C5a and C5a-d-Arg, we use an N-terminal fusion of Thioredoxin (TrxA), which is a 222 small (16 kDa), soluble, and thermostable protein50,51, and we reasoned that a complex of Trx-223 C5a/C5a-d-Arg-C5aR2 may help structural analysis using cryo -EM. Therefore, we first 224 measured the pharmacology of Trx -C5a/C5a-d-Arg vis-à-vis C5a/C5a -d-Arg, and observed that 225 they were equally potent and efficacious in βarr recruitment assay ( Figure S3B). Next, we 226 reconstituted Trx -C5a-C5aR2 and Trx -C5a-d-Arg-C5aR2 complexes and subjected them to 227 cryo-EM analysis. While both complexes exhibited 2D class averages with clear density for 228 the ligand and the receptor, Trx -C5a-d-Arg-C5aR2 complex ( Figure S3C ) yielded a low -229 resolution 3D reconstruction while C5a -C5aR2 complex yielded a structure at an overall 230 resolution of 3.8 Å (Figure 3C and S4A). 231 The C5a -C5aR2 structure exhibits the canonical 7TM architecture with the 2 nd 232 extracellular loop (ECL2) adopting an anti -parallel β -hairpin conformation as previously 233 observed in the structures of C5aR111 (Figure 3C). Despite moderate resolution, the cryo-EM 234 map allowed unambiguous modelling of the transmembrane region, ECLs and ICLs, and C5a 235 (Figure S6A). In addition, clear density for the distal N -terminus of the receptor from Pro20 236 and helix 8 are also observed in the structure ( Figure 3C and S8). Although a structure of 237 C5aR2 in a prototypical inactive state is not available, the comparison with antagonist-bound 238 C5aR1 structure published previously 52 (PDB: 6C1R), reveals significant conformational 239 changes reminiscent of an active receptor conformation. For example, TM6 of C5aR2 shifts 240 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 10 outward by ~6.5 Å (as determined with respect to the Cα positions of Leu3276.34 in C5aR1 and 241 Arg2326.34 in C5aR2), while TM7 moves inward by ~4 Å (measured from the Cα atoms of 242 Gly3047.57 in C5aR1 and Gly2957.57 in C5aR2) (Figure 3D). Moreover, the comparison of C5a-243 C5aR2 structure with that of C5a-C5aR1 shows an overall similar conformation with a main -244 chain RMSD of ~1 Å (Figure 3E). In the inactive C5aR1 structure, helix 8 assumes an inverted 245 orientation, and reorients itself significantly upon receptor activation11,52. The position of helix 246 8 in C5a-C5aR2 structure is also reminiscent of the active state C5aR1 (Figure 3D). 247 The core domain of C5a interacts with the N -terminus of C5aR2 while the carboxyl -248 terminus engages with the orthosteric binding pocket representing a two -site binding mode, 249 similar to that of C5aR1 ( Figure 3F). The extensive interaction between C5aR2 N -terminus 250 and C5a core domain, a defining characteristic of the C5a-C5aR2 structure, involves several 251 non-bonded contacts including the interaction of K20, D24, and I41 of C5a with Val21, Leu24, 252 and Asp25 of C5aR2, respectively (Figure S3D). Interestingly however, there are also notable 253 differences between C5a -binding to C5aR1 and C5aR2 ( Figure 3G-I and S3D -F). For 254 example, the N-terminus of C5aR1 is oriented to engage with the loop between helix 2 and 255 helix 3 (H2-H3 loop) of C5a, while the N-terminus of C5aR2 is shifted such that it interacts with 256 H2 as well as H3-H4 loop of C5a (Figure 3E and S3D-E). While ECL2 of C5aR1 interacts with 257 the H1 residues of C5a, in C5aR2, ECL2 is displaced towards the extracellular opening of the 258 orthosteric pocket and positions near to the H2 -H3 loop in the C5a-C5aR2 structure (Figure 259 3E and S3F). These observations are further corroborated by site-directed mutagenesis data 260 on C5aR1 as discussed previously42, and C5aR2 here (Figure S3G-H). For example, Arg5.42 261 is conserved in both C5aR1 and C5aR2 but makes contact with C5a only in C5aR1. 262 Accordingly, its mutation to alanine leads to a dramatic loss of βarr1 recruitment for C5aR1 263 (Figure 2C) while it remains unchanged for C5aR2 (Figure S3G). Interestingly, the pattern of 264 βarr recruitment upon C5a and C5a-d-Arg stimulation of a set of C5aR2 mutants reflect a near-265 identical response, suggesting a conserved mode of binding of these two ligands to C5aR2 266 leading to a similar potency and efficacy as mentioned earlier (Figure 3B and S3G). 267 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 11 In order to validate the structural observations further, we performed HDX time-kinetics 268 analyses on apo -C5aR2 and C5a -C5aR2 complexes. We observed a robust sequence 269 coverage of C5a and C5aR2 in these experiments as outlined together with the technical 270 details in Figure S 9A-B. Upon C5a binding, the HDX levels of several regions in C5aR2 271 showed a decrease including the N -terminus, ECL3, TM4, TM6, TM7, and ICL2 ( Figure 3J, 272 S9C and Supplementary Dataset 1B, 1C, 1E and 1F). This pattern is in sync with the cryo-273 EM structure such as a direct interaction of C5a with the N -terminus and ECL2 region. In 274 addition, the observed reduction in HDX levels in the TM regions suggest possible activation-275 dependent conformational changes through allosteric mechanism, which is also corroborated 276 by the structural comparison of C5a -C5aR2 structure with the antagonist PMX53 -bound 277 inactive state of C5aR152 (PDB: 6C1R). Furthermore, the decrease in HDX levels at the ICL2 278 and the distal end of TM7 also likely reflect agonist -induced conformational propagation 279 resulting in a short helix formation in ICL2 and reorientation of helix 8 upon activation. Finally, 280 the comparison of HDX level of C5a in the free and receptor-bound state reveals a significant 281 decrease in both, the carboxyl-terminal and amino-terminal regions, which aligns well with the 282 two-site binding mode of C5a on C5aR2 (Figure 3J and Figure S9D). 283 Structural basis of C5a-d-Arg-recognition by C5aR2 284 Our functional characterisation of mouse C5aR2 for βarr1/2 recruitment in HEK -293 cells 285 shows near identical response for mC5a and mC5a -d-Arg as observed for hC5a and hC5a-d-Arg 286 on hC5aR2 (Figure 3B). We reasoned that mC5a -d-Arg-mC5aR2 may yield a complex better 287 amenable to structural analysis compared to the human receptor, and thereby, help us in 288 decipher the binding mode of C5a -d-Arg on C5aR2. Interestingly, in our attempt to purify 289 mC5aR2, we observed that unlike hC5aR2, it exhibits a distinct and substantial dimeric 290 population (Figure 4A), and it allowed us to determine the structures of mC5a -d-Arg-bound 291 mC5aR2 complex as a dimer using cryo-EM. As anticipated, structure determination revealed 292 dimeric assembly of mC5aR2 wherein both the protomers lie adjacent to each other in a single 293 detergent micelle bound to mC5a-d-Arg (Figure 4B, Figure S4B, S6B and S8). The mC5a-d-Arg-294 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 12 mC5aR2 exhibits the canonical 7TM architecture with the 2 nd extracellular loop (ECL2) 295 adopting an anti -parallel β-hairpin conformation as previously observed in the structures of 296 C5aR1 ( Figure 4B). The structural superposition of hC5aR2 and one of the protomer of 297 mC5aR2 showed a comparable RMSD values for TM regions while TM1 and cytoplasmic 298 loops and helix 8 displayed significant deviation (Figure 4C). Moreover, our structural analysis 299 uncovered a previously unrecognised dimeric assembly in mouse C5aR2, which is stabilised 300 by the two inter-protomer disulfide bonds between Cys306 and Cys313 in helix 8 of opposing 301 protomers. This is further held together by tight hydrophobic packing between Phe 1.43 and 302 Leu1.44 of TM1 ( Figure 4D). To our knowledge, this represents the first report of a dual 303 disulfide-mediated covalent linkage stabilising a class A GPCR dimer. The presence of this 304 covalent interface exclusively in mouse C5aR2 suggests a species -specific structural 305 adaptation that may have significant functional consequences compared to human receptor. 306 This finding broadens the current understanding of GPCR dimerization and also underscores 307 the significance of species-specific receptor functions. Similar to C5a, C5a-d-Arg also displays 308 a two-site binding mode on C5aR2, acquires an overall similar positioning as C5a with slight 309 shift in the core-domain (Figure 4E). This is also reflected in identical positioning of terminal 310 residues i.e., R74 and G76 of C5a and C5a -d-Arg, respectively (Figure 4F). A c omparative 311 analysis of ligand-receptor interactions reveals structural features that may contribute to the 312 retained potency of C5a -d-Arg at C5aR2. The binding of C5a -d-Arg to mC5aR2 is characteri zed 313 by a n extensive interaction, wherein G76 aligns in the position of R74 in C5a and forms 314 hydrogen bonds with Arg1794.64 and the backbone nitrogen of Arg2105.42 (Figure 4F and 4G). 315 The C-terminal region engages in multiple additional interactions, for example, K71 forms salt 316 bridge with Glu5.35 and hydrogen bond Ile6.58, H70 forms salt-bridge with Gly193ECL2, Q74 and 317 L75 forms hydrogen bonds with Glu 7.35 and Arg6.55. This results in a network comprising ten 318 hydrogen bonds and one salt -bridge distributes across C -terminal residues ( Figure 4G). 319 Taken together with the structural analysis of C5a -d-Arg-C5aR1, these observations suggest 320 that while C5aR1 may rely more critically on R74 -mediated contacts for conformational 321 stabilisation linked to βarr signalling, C5aR2 can accommodate R74 loss by redistributing 322 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 13 interactions to alternative residues in the C -terminal region, thereby maintaining receptor 323 activation to an efficacy similar to that of C5a. 324 Discovery of a C5aR2-selective agonist 325 Considering the similarities between the interaction of C5a with C5aR1 and C5aR2 in the 326 orthosteric pocket, we envisioned that peptides derived from the carboxyl -terminus of C5a 327 may also serve as C5aR2 agonists (Figure 5A). Accordingly, we screened a broad set of C5a- 328 and C3a-derived peptides on C5aR2 in βarr recruitment assay ( Figure 5B and S 10A) and 329 observed that C5a pep and EP54 exhibited a significant response ( Figure 5B and S10E-F). 330 However, these peptides are also known to activate C3aR and C5aR1 11, and previously 331 described C5aR2-selective peptides, i.e., P32 and P59, have rather low efficacy (Figure 5B). 332 Therefore, we revisited our previous data focused on the identification of selective peptide 333 agonists for C5aR153, and we identified two peptides namely, BM2020 -7 and BM2020-8 that 334 had improved efficacy and potency at C5aR2 compared to P32 and P59 54 (Table S1 ). 335 However, neither peptide was selective for C5aR2 with BM2020-7 having moderate activity at 336 C3aR and BM2020-8 being a potent agonist of both C3aR and C5aR1. Considering that C5a 337 and C5a-d-Arg have similar efficacy at C5aR2, we hypothesised that we could modify the C -338 terminal arginine of BM2020 -7 and BM2020 -8 to introduce selectivity for C5aR2. This 339 hypothesis is also substantiated by our previous observation that the terminal arginine in these 340 peptides is crucial for their potency at C3aR and C5aR111. 341 Therefore, we synthesised a set of BM2020 -7 analogues where we systematically 342 modified the C -terminal residue to asparagine, aspartic acid, glutamine, glutamic acid, 343 histidine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or serine. We then assessed the 344 ability of these peptides to activate C5aR2 -mediated βarr2 recruitment at a concentration of 345 100 µM. Of these peptides, the tyrosine analogue demonstrated full agonist activity (relative 346 to C5a) at C5aR2 ( Figure S 10B) and subsequent concentration -response experiment 347 revealed that it had an EC 50 of ~0.9 μM (Figure S10C). As predicted, this tyrosine analogue 348 did not activate C3aR or C5aR1-mediated ERK phosphorylation up to 100 µM (Table S1). As 349 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 14 BM2020-8 was a more potent, albeit non-selective, agonist of C5aR2 compared to BM2020-350 7, we replaced the arginine in BM2020 -8 with tyrosine to produce [Tyr8]BM2020-8, which had 351 an EC50 of 35 nM at C5aR2 (Figure 5C) but was a partial agonist at C5aR1 (EC50 = ~120 nM, 352 41% efficacy relative to C5a ). Finally, we replaced the phenylalanine at position 1 in 353 [Tyr8]BM2020-8 with tyrosine to generate [Tyr1, Tyr8]BM2020-8, referred to as R8Y hereon, which 354 remained a full agonist of C5aR2 (EC 50 = ~13 nM) (Figure 5C), and it did not activate C3aR 355 or C5aR1 up to 10 µM (Figure S 10D and Table S1 ). Finally, we confirmed the subtype 356 selectivity of R8Y in HEK -293 cells expressing comparable levels of C3aR, C5aR1, and 357 C5aR2 (Figure 5D and S10G). 358 Molecular basis of C5aR2 activation by peptide agonists 359 In order to understand the molecular basis of subtype -selectivity and activation of C5aR2 by 360 the peptide agonists, we focused our efforts on determining the structures of C5aR 2 in 361 complex with EP54 (C3aR/C5aR1/C5aR2 cross -reactive), C5a pep (C5aR1/C5aR2 cross -362 reactive), and R8Y (C5aR2 selective). As the strategy used for C5a -C5aR2 is not feasible 363 here, we tested a previously described anti-C5aR2 monoclonal antibody, referred to as 4C855, 364 as a fiducial marker for cryo-EM analysis (Figure S11A). We first confirmed the ability of this 365 antibody to recognize C5aR2 and observed that it effectively blocks C5a -induced βarr 366 recruitment at C5aR2 but not at C5aR1 or C3a-induced βarr recruitment at C3aR (Figure 5F 367 and S10L). We also did not observe any agonistic effect of 4C8 by itself, and therefore, taken 368 together, these data confirm that 4C8 acts as a competitive and selective inhibitor of C5a at 369 C5aR2. Interestingly however, 4C8 pre -incubation did not impact βarr recruitment at C5aR2 370 in response to any of the peptide agonists suggesting that binding of 4C8 is permissive for 371 peptide interaction and presumably receptor activation (Figure 5F and S10L). 372 Next, we generated the Fab version of 4C8 using papain digestion and subsequent 373 purification, and observed that it formed a stable complex with C5aR2 either in the apo -state 374 or in presence of peptide agonists (Figure S11B-E). We successfully determined the cryo-EM 375 structures of Fab4C8-stabilized apo-C5aR2, EP54-hC5aR2, C5apep-C5aR2, and R8Y-C5aR2 376 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 15 complexes at 3.0 Å - 3.2 Å resolution range (Figure S4C and S5A-C), and the cryo-EM maps 377 allowed unambiguous modelling of the receptor regions and the ligands (Figure 5G and S7A-378 D). Structural alignment of these structures reveals an overall similar conformation, with an 379 RMSD of <1 Å across the main -chain atoms ( Figure 6A). A complete description of the 380 residues that are resolved is presented in Figure S 8. Expectedly, unlike the C5a -C5aR2 381 structure, the N-terminus of the receptor was not resolved well in these structures owing to a 382 lack of interaction with the peptide agonists, which are likely engaged only with the orthosteric 383 binding pocket. In addition, the carboxyl-terminus of the receptor including helix 8, and some 384 of the ICL2 and ICL3 loops were also not resolved well, indicating their structural flexibility 385 (Figure S8). 386 In addition to the N -terminus, the apo -C5aR2 structure exhibits notable flexibility in 387 other regions as well when compared to the C5a-C5aR2 structure (Figure S12A). Specifically, 388 the loop connecting β -strand 1 and β -strand 2 of ECL2 (G178 –R183) shifts away from the 389 orthosteric binding pocket, likely due to the absence of stabilizing interactions with the core 390 domain of C5a (Figure S12B). Likewise, the extracellular end of TM6 and TM7, and the ECL3, 391 exhibit an outward movement in the apo -C5aR2 structure compared to C5a -C5aR2 (Figure 392 S12A-B). This structural rearrangement is supported by an increase in the HDX level in this 393 region in apo - vs. C5a -bound states of C5aR2 (Figure 3J ). The intracellular side of the 394 receptor shows an overall smaller cavity in the apo-C5aR2 structure with an approximate 395 volume of ~ 3500 Å 3 compared to that of C5a-C5aR2 with a volume of ~42 00 Å 3. This 396 difference likely originates from distinct positioning of the TM helices on the intracellular side 397 in the apo-C5aR2, which emulates an inactive-like conformation of receptor with reference to 398 the antagonist-bound C5aR1 structure52 (PDB: 6C1R) (Figure S12B). 399 Reminiscent of the binding mode of C5a, the peptide agonists EP54, C5apep and R8Y 400 also adopt a hook -like conformation and penetrate deep into the orthosteric pocket of the 401 receptor at a vertical distance of ~8 -9 Å from the conserved Trp246 6.48, and make extensive 402 interactions with the residues from TM2-7, ECL1 and ECL2 (Figure 6B). In accordance with 403 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 16 the likely dispensable role of the terminal arginine (R74) of C5a in C5aR2 activation as 404 reflected by C5a-d-Arg pharmacology, the terminal residues of C5apep (i.e., d-Arginine) and R8Y 405 (tyrosine) engage via either hydrogen bonds or polar networks with Glu2737.35, Tyr2496.51and 406 Arg1734.64, similar to that observed for C5a and EP54 (Figure 6C-D). A comparison of overall 407 interactions for the peptide agonists and C5a revealed a set of residues common to all the 408 agonists, located either within the orthosteric pocket, or in the ECL2, suggesting a convergent 409 mode of ligand recognition (Figure 6E). On the other hand, C5a engages with a few additional 410 unique residues, particularly on ECL3 and the N -terminus, which likely imparts a higher 411 potency for the receptors, analogous to that observed for C5aR1 as well. 412 Structural analysis of C5a pep-C5aR2 structure reveals that C5a pep forms seven 413 hydrogen bonds along with several non -bonded contacts within the orthosteric pocket of 414 C5aR2. The terminal DAR6 in C5apep forms ionic bond and cation-π interaction with Tyr2496.51 415 and hydrogen bonds with Arg1734.64 and Arg2045.42, while it engages with Tyr119 3.37 through 416 ionic interaction (Figure 6D). In addition, other non -bonded contacts that maintain the 417 conformation of DAR6 in C5a pep include contacts with Ser169 4.60 and Gly253 6.55 of C5aR2 418 (Figure S12C). K2 in C5apep engages with Val188ECL2 and Glu2737.35 through a hydrogen bond 419 and with Val187 ECL2 through a non -bonded contact while MEA1 in C5a pep makes a π -π 420 interaction with His176ECL2 and engages with Val187ECL2 and Asp189ECL2 through non-bonded 421 contacts to further facilitate the stabilization of the ligand within the orthosteric pocket. (Figure 422 S12C). 423 Further, we compared the binding modes of C5a pep on C5aR1 and C5aR2 using the 424 structures presented here with those reported recently 42. We observed that, although the 425 overall backbone conformation of C5apep in the C5apep-C5aR2 structure closely resembles with 426 that observed in C5apep-C5aR1 complex, there is a notable difference in the orientation of the 427 terminal DAR6 residue between the two receptors (Figure 6F). In the C5apep-C5aR2 structure, 428 the guanidinium group of the DAR6 residue undergoes a linear horizontal shift of ~5 Å 429 towards TM5 within the orthosteric pocket compared to C5a pep-C5aR1. Furthermore, the 430 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 17 orientation of the MEA1 and K6 in C5apep between the C5aR1 and C5aR2 are also distinct as 431 they are positioned opposite to each other. Despite these differences, comparing the overall 432 interaction of C5apep with C5aR1 and C5aR2 reveals a similar interaction pattern, as expected 433 from its ability to activate both C5aR1 and C5aR2 (Figure 6G). 434 Even though C5a pep, EP54 and R8Y exhibit a similar binding mode on C5aR2, there 435 are clear differences while comparing the intricate details of the binding of these three peptide 436 agonists at C5aR2, as well as when comparing EP54 binding to C5aR1 vs. C5aR2, and R8Y 437 vs. C5a binding to C5aR2. For example, compared to the positioning of the guanidino group 438 of the terminal arginine of EP54 in the EP54-hC5aR1 structure42, the terminal Arg10 of EP54 439 in the EP54-C5aR2 structure appears to be slightly constrained, and docks into an alternative 440 sub-pocket within the orthosteric site ( Figure 6H). Moreover, in C5aR2, EP54 is stabilized 441 through the formation of hydrogen bonds with Glu273 7.35 and Val188 ECL2, and an ionic 442 interaction with the hydroxyl group of Tyr249 6.51(Figure 6D). Similar to C5a pep, several other 443 residues of EP54 establish non -bonded contacts within the orthosteric binding pocket of 444 C5aR2 (Figure S12D). Despite these subtle differences, a global interaction analysis of EP54 445 on C3aR, C5aR1 and C5aR2 revealed a conserved binding mechanism by the involvement of 446 similar residues lining orthosteric binding pocket and ECL2, perhaps attributing to the agonistic 447 property of EP54 across complement receptors (Figure 6I). 448 In the R8Y-C5aR2 structure, Y8 of R8Y undergoes a linear transition downwards by 449 ~5.5 Å relative to the spatial positioning of DAR6 in C5a pep-C5aR2, and it engages with 450 Tyr2496.51 through a π -π interaction and with Arg204 5.42 through a salt bridge ( Figure 6D). 451 Additionally, K2 of R8Y establishes hydrogen bonds with Val188ECL2 and Leu2566.58, while the 452 backbone oxygen of G5 in R8Y forms hydrogen bonds with Arg173 4.64 (Figure S1 2E). 453 However, R74 of C5a in C5a-C5aR2 occupies the same position as Y8 of R8Y and establishes 454 a hydrogen bond with Glu273 7.35, while K68 of C5a forms three hydrogen bonds, one each 455 with Val188 ECL2, Glu197 5.35 and Thr257 6.59 (Figure S12F). These additional interactions by 456 C5a compared to R8Y, together with the involvement of N -terminus of the receptor, possibly 457 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 18 explains the relatively weaker potency of R8Y at C5aR2 compared to C5a. Finally, a structural 458 alignment of ligand -receptor interactions for natural and synthetic -peptide agonist -bound 459 structures of C3aR, C5aR1, and C5aR2 reveal that C5aR2 also employs a “Five-Point-Switch” 460 to recognize diverse ligands ( Figure 6J), as identified for C3aR and C5aR1 in the recent 461 study42. 462 Species-specific pharmacology and transducer-coupling of C5aR2 463 Inspired by the dramatic species-specific pharmacology observed at the C3aR and C5aR142, 464 we also probed the activity of the C5aR2-selective agonist discovered here i.e. R8Y. Strikingly, 465 our functional assays revealed remarkable selectivity of R8Y for human C5aR2, and it does 466 not seem to activate mouse C5aR2 as assessed using βarr recruitment assay (Figure 5E and 467 S10I and S1 0K). In addition, the sequence comparison of the human and mouse C5aR2 468 shows a different phosphorylation signature in the carboxyl -terminus wherein the mouse 469 receptor contains the P-X-P-P motif critical for driving βarr recruitment and activation while the 470 human receptor does not (Figure S13A). This is further reflected in the pattern of reactivity of 471 Ib30, an intrabody sensor designed to report an active conformation of βarr1 in cellular 472 context56, wherein we observe a robust signal of Ib30 reactivity for the mouse receptor but not 473 for the human C5aR2 ( Figure S13B-C). These data further corroborate the species -specific 474 specialization of transducer-coupling at C5aR2, which converges to a similar observation for 475 C5aR1 in terms of βarr interaction and activation as reported recently42. 476 Molecular insights into βarr-bias encoded at C5aR2 477 Comparison of the C5a -C5aR2 structure with C5a -C5aR1 complex (PDB: 8IA2) reveals a 478 cytoplasmic pocket dimension of ~30 Å (as measured from the Cα atoms of Trp141 ICL2 to 479 Phe2917.53) in C5aR2 with a pocket volume of 4,250 Å3 while in C5aR1, the pocket spans ~22 480 Å (as measured from the Cα atoms of Cys144ICL2 to Tyr3007.53) with a pocket volume of ~2,900 481 Å3. Thus, the cytoplasmic pocket of C5aR2 appears to be relatively wider, and in addition, it is 482 also less charged and more hydrophobic compared to that of C5aR1. These differences may 483 prevent a stable docking, and conformational changes required for an efficient coupling and 484 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 19 activation of G-proteins to C5aR2 (Figure 7A). The polar and charged amino acids within the 485 cytoplasmic pocket in the C5aR1 structure enable strong electrostatic and hydrogen -bond 486 interactions with G-proteins, thereby stabilizing the complex and facilitating activation (Figure 487 7A). However, a significant variation in these key polar and charged residues can be observed 488 in C5aR2, which instead has a more hydrophobic environment. These altered pocket 489 properties are ill-suited to efficiently interact with the largely hydrophilic and charged surfaces 490 of G-proteins, resulting in an absence of G-protein-coupling (Figure S14A). 491 While ICL3 and helix 8 were unresolved in the C5aR2 complexes with peptide 492 agonists, they are well resolved in the C5a -C5aR2 complex ( Figure S 8). Structural 493 comparison with the C5a -C5aR1 complex revealed that TM 6 in C5aR2 is shorte ned by five 494 residues, TM5 is shortened by three residues, and the ICL3 region in the C5a-C5aR2 structure 495 is also shortened (Figure 7B). Previous studies suggest that TM5 is structurally important for 496 maintaining the integrity of the G -protein-binding interface. Shortening of TM5 or altering its 497 structure can disrupt the proper positioning of ICL3 and the cytoplasmic cavity, impairing G -498 protein-coupling40,57,58. Furthermore, residues of ICL3 and helix-8 have been found critical for 499 the engagement of GPCRs with the α5 helix of G-proteins59-67 (Figure 7B and S14B). Dynamic 500 behaviour and potentially occlusive conformational states of ICL3 and helix 8 in C5aR2 are 501 likely to further contribute to hindering the formation of a proper cytoplasmic pocket required 502 for the docking of α5 helix in C5aR2. 503 The residues corresponding to ICL2 of GPCRs typically adopt a short α -helical turn 504 and contribute to the GPCR -G-protein interface by interacting with a hydrophobic groove 505 formed by α5, αN, β1 and β3 strands of the Gα subunit enhancing binding stability66,68-70. The 506 ICL2 residues in the R8Y-, EP54-, and C5apep-bound C5aR2 structures adopt a linearized loop 507 conformation and undergo a linear shift away from the receptor -G-protein interface and also 508 the core of the receptor ( Figure 7C). Although, the ICL2 residues form a half -helical turn in 509 the C5a-C5aR2 structure, their orientation still remains consistent with the peptide agonist -510 bound C5aR2, positioning them away from both, the receptor core and the G-proteins (Figure 511 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 20 7C). In addition, the presence of a small residue at the end of TM3 (Gly1383.56) and proline as 512 the first residue of ICL2 (Pro139ICL2) prevent the formation of a kink in ICL2, which in turn limits 513 the ability of ICL2 to adopt a helical conformation, further restraining the engagement with 514 G-proteins71 (Figure 7C). Therefore, the lack of a kink and the loss of a helical conformation 515 in ICL2 likely alter the spatial positioning of the corresponding residues, thereby weakening 516 the hydrophobic contacts with the α5 helix and polar interactions with the αN helix in G -517 proteins. 518 Finally, two of the conserved GPCR motifs namely the DRY motif in TM3 and the 519 NPxxY motif in TM7 display an altered sequence in C5aR2, i.e., DLC and NPxxF, respectively. 520 Interestingly, Cys1333.53 (TM3) in the DLC motif forms a disulfide bond with Cys2195.57 in TM5, 521 which in turn rigidifies and restrains the movement of TM3 and TM5 in the receptor upon 522 activation (Figure 7D). Moreover, in C5a -C5aR1, Arg1343.50 in the DRY motif interacts with 523 Tyr3007.53 of the NPxxY motif in the active state, which simultaneously engages through an 524 ionic bond with the Cys351 of α5 helix in the G -proteins. In contrast, the Phe300 7.53 of the 525 NPxxF motif in C5aR2 cannot interact with Leu1323.50 of the DLC motif due to the shorter side 526 chain of leucine, and which in turn results in a more flexible cytoplasmic pocket that is 527 inefficient for effective G-protein-coupling (Figure 7D). Taken together, the hydrophobicity of 528 the binding cavity, shortening of TM5 and TM6, and the unique DLC motif further contribute to 529 the inability of C5aR2 to couple with, and activate, G-proteins (Figure 7E-F). 530 Intrigued by the remarkable differences at the cytoplasmic interface of C5aR1 and 531 C5aR2, we hypothesised that substituting the cytoplasmic half of C5aR2 with that of C5aR1 532 might impart G -protein coupling ability. Based on this, we designed chimeric C5aR1 and 533 C5aR2 constructs in which cytoplasmic halves were reciprocally swapped and subsequently, 534 measured G -protein activation ( Figure 7E). As anticipated , the C5aR2 -C5aR1 chimera, 535 harboring cytoplasmic interface of C5aR1, displayed robust G-protein dissociation and cAMP 536 responses, with only slight shifts in potency and efficacy. This represents first demonstration 537 of a functional gain of G -protein coupling in any ACR. Conversely, the C5aR1 construct 538 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 21 bearing the cytoplasmic interface of C5aR2 completely lost G-protein activation, mirroring the 539 behaviour of native C5aR2. Together, these results indicate that the molecular determinants 540 underlying the intrinsic signaling bias of C5aR2 are predominantly localized within its 541 cytoplasmic interface. Collectively, these structural constraints spanning the hydrophobic 542 cavity, shortened TM5 and TM6, and the unique DLC motif, locks C5aR2 into a G -protein 543 incompetent state (Figure 8A). 544

545 The generation of C3a-d-Arg and C5a-d-Arg is typically conceived as a regulatory mechanism to 546 dampen the excessive inflammatory response, which is believed to arise from a significantly 547 reduced affinity to their corresponding receptors 31-33,43,44,72. However, our findings presented 548 here, suggest a functional specialization instead, with the three anaphylatoxin receptors 549 perceiving the impact differently. While C3a -d-Arg exhibits near -complete loss of inducing 550 G-protein and βarr -coupling to C3aR, C5a -d-Arg maintains nearly identical activation of 551 G-protein-coupling at C5aR1, but significantly attenuated βarr response. On the other hand, 552 C5a-d-Arg is able to activate βarr -coupling at C5aR2 at levels indistinguishable from C5a. 553 Considering that C5aR2 is weaker than C5aR1 in terms of overall βarr recruitment and 554 completely lacks G -protein-coupling, our data seem to suggest that C5a -d-Arg is encoded to 555 minimize βarr response through C5aR1 and C5aR2 instead of abrogating transducer-coupling 556 entirely. Thus, it is tempting to speculate that G -protein-mediated responses are linked to 557 desirable downstream outcomes and hence maintained via C5aR1, while sustained βarr-558 mediated responses may be deleterious and hence, minimized via both C5aR1 and C5aR2. 559 In fact, our neutrophil mobilization data in mice where C5a -d-Arg is significantly weaker than 560 C5a, supports such a possibility since excessive C5a-mediated neutrophil mobilization is 561 linked with tissue and organ damage 73-75. It is also possible that attenuated βarr recruitment 562 at C5aR1 is designed to sustain G-protein signaling via C5aR1, as receptor desensitization 563 through βarrs in response to C5a results in rapid blunting of downstream responses. Probing 564 these intriguing possibilities in future studies should help illuminate the correlation of the 565 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 22 naturally-encoded biased -signaling with functional outcomes in physiological and 566 pathophysiological context. 567 As discussed previously, we have recently observed a striking specialization at the 568 level of species-specific pharmacology of the C3aR and C5aR142. Along the same lines, the 569 C5aR2-selective agonist identified here, also exhibits a strong preference for the human 570 receptor compared to mouse C5aR2. Our previous study has successfully demonstrated that 571 structure-guided receptor mutagenesis can help reverse the species -specific pharmacology, 572 for example, for C5a pep at C5aR1 11. Therefore, it would be interesting to employ a similar 573 approach to design gain -of-function variants of R8Y that work on mouse C5aR2, and such 574 ligands may help dissect the functional separation of the two receptors in mouse model. In a 575 broader sense, these emerging indications that the human and mouse receptors may have 576 dramatically different pharmacology for natural and synthetic ligands, call for a more careful 577 integration of the concept of species -specific pharmacology in drug discovery campaigns to 578 reduce the disconnect between in vitro data and pre-clinical studies. 579 It is also worth noting that the cryo-EM structures of CXCR7 (ACKR3), which is also a 580 βarr-biased 7TMR, have also been reported recently 71,76, and the structural interpretation in 581 terms of conformational dynamics of the cytoplasmic interface leading to inefficient G-protein 582 interaction aligns with that of C5aR2 reported here although there are some key differences 583 as well. For example, unlike C5aR2, CXCR7 harbors conserved DRY and NPXXY motifs but 584 still fails to activate G -proteins, and therefore, it is likely that additional mechanisms specific 585 to CXCR7 may also exist rendering it incapable of coupling to G -proteins. Finally, the Duffy 586 antigen receptor for chemokines (DARC), also known as ACKR1, lacks any measurable 587 coupling to either G -proteins or βarrs, which is attributed primarily to shortened TM5 and 6, 588 and a kink at the cytoplasmic portion of TM3 40, which is significantly different from that 589 observed in C5aR2. 590 There are still several interesting questions that remain to be answered in the context 591 of ACRs. For example, do the ACRs converge to a common signaling pathway mediated via 592 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 23 βarrs? For prototypical GPCRs, agonist -induced activation of ERK1/2 phosphorylation has 593 been used as a quintessential and convergent readout of signaling 77,78, however, the ACR 594 activation does not appear to effectively elicit this pathway. While future studies focused on 595 deciphering the signaling networks at these receptors will illuminate the downstream signaling 596 aspect further, it is worth noting that there are receptor-specific differences, even at the level 597 of βarr-coupling profile of these ACRs. For example, ACKR2 -4 appears to show an agonist-598 dependent βarr -recruitment while GPR182 (ACKR5) appears to have a constitutive βarr 599 recruitment that is even proposed to decrease in response to selected ligands 12,79,80. The 600 molecular mechanisms underlying these differences need further exploration at the level of 601 receptor conformation and phosphorylation in cellular context. We also note that structural 602 snapshots provide a static image of the molecules, and the receptor -transducer coupling is 603 likely to be regulated in a more dynamic fashion. Therefore, additional orthogonal approaches 604 to probe the dynamic activation and conformational changes of ACRs may shed additional 605 light in their lack of interaction with G -proteins. Taken together, these observations suggest 606 possible specialization at the level of receptor -specific mechanisms leading to their diverse 607 functional outcomes despite an overall converging thematic connection. 608 In conclusion, our study elucidates the molecular basis of naturally -encoded ligand-609 bias and receptor-bias at the complement anaphylatoxin receptors, identifies the first-in-class, 610 C5aR2-selective peptide agonist, and guides the design of signaling-biased C5a variants. Our 611 findings have direct and broad implications for understanding the framework of biased 612 agonism at 7TMRs with direct implications for better therapeutic design. 613 Declaration of interest 614 Authors declare no competing interests. 615

616 Research on complement anaphylatoxin receptors in A.K.S.’s laboratory is currently 617 supported by the Senior Fellowship of the DBT Wellcome Trust India Alliance 618 (IA/S/20/1/504916), the Indian Council of Medical research (EMDR/SG/14/2024 -01-02127), 619 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 24 and the Department of Science and Technology (DST/TTI/TC/AMR/COE/2023/5). Part of the 620 cryo-EM data was collected at the National cryo-EM Facility at IIT Kanpur established with the 621 support from ANRF/SERB (IPA/2020/000405). A.K.S. is the Sonu Agrawal Memorial Chair 622 Professor. This research was also supported by the JSPS KAKENHI grant numbers 21H05037 623 (O.N.) and 23KJ0491 (F.K.S.), the Platform Project for Supporting Drug Discovery and Life 624 Science Research (Basis for Supporting Innovative Drug Discovery and Life Science 625 Research [BINDS]) from the Japan Agency for Medical Research and Development (AMED) 626 grant numbers JP22ama121012 and JP22ama121002 (O.N.). A.I. was funded by KAKENHI 627 JP21H04791 and JP24K21281 from the Japan Society for the Promotion of Science (JSPS); 628 JP22ama121038 and JP22zf0127007 from the Japan Agency for Medical Research and 629 Development (AMED); JPMJFR215T and JPMJMS2023 from the Japan Science and 630 Technology Agency (JST); The Uehara Memorial Foundation. Research in T.M.W.’s 631 laboratory is supported by the National Health and Medical Research Council (APP2009957) 632 and R.J.C.’s research is supported by the National Health and Medical Research Council 633 (APP2012661). HDX-MS work in K.Y.C.’s laboratory was supported by grants from the 634 National Research Foundation of Korea funded by the Korean government (NRF -635 2021R1A2C3003518 and NRF -2019R1A5A2027340 to K.Y.C.) . We also acknowledge 636 Australian Red Cross Lifeblood and human donors for providing blood for our research. We 637 also thank Kayo Sato, Shigeko Nakano and Ayumi Inoue in the Inoue lab for their assistance 638 in the plasmid construction and the NanoBiT assay. We sincerely thank Dr. Charles Mackay 639 and Caroline Ang for providing the 1D9 and 4C8 hybridoma clones. We thank Shachie Sinha 640 for helping with cellular assays, and Ashna Reyaz, Calvin D’Souza, and Debdatta Mukherjee 641 with protein purification. 642 Authors’ contribution 643 DT and MKY expressed and purified the receptor and prepared the complexes with help from 644 AD; DT expressed and purified 4C8 antibody and prepared the Fab with help from NR in the 645 early stages; KS and FKS screened the samples for cryo -EM, collected and processed the 646 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 25 data with help from KY, HSO, KH, and built the initial model under the supervision of ON; AD 647 and SM carried out the functional assays in HEK -293 cells; XXL carried out the Ca 2+ flux, 648 pERK, and BRET assays, and experiments in primary HMDMs/BMDM/PMNs, under the 649 supervision of TMW; CSC carried out the in vivo neutrophil mobilisation assay with help from 650 JNF and TL under the supervision of JDL and TMW. JCD identified and characterized R8Y 651 under the supervision of RC and TMW; KK and D A performed and analyzed the HDX 652 experiments under the supervision of KYC; NB expressed and purified wild-type C5a and C5a-653 d-Arg; TMS performed the MD simulation studies under the supervision of JS; AI carried out the 654 C5aR1-GRK interaction assay; RB and MKG also processed cryo-EM data of apo-C5aR2 and 655 R8Y-C5aR2 collected at IITK, refined and built the final models for all the cryo-EM structures, 656 carried out the analyses, and helped prepare the figures together with DT and NR; RC, KYC, 657 RB, FKS, TMW, ON, and AKS supervised the overall project. All authors contributed to writing 658 and editing the manuscript. 659 Data availability 660 The cryo-EM maps and structures have been deposited in the EMDB and PDB with accession 661 numbers PDB ID- 9V3C, EMD-64752 (Trx-C5a-C5aR2), PDB ID- 9WDI, EMD- 65890 (mC5a-662 d-Arg-mC5aR2), PDB ID- 9V35 , EMD - 64749 (Fab4C8-Apo-C5aR2), PDB ID- 9V3Y, EMD- 663 64761 ( Fab4C8-C5apep-C5aR2), PDB ID - 9V38, EMD - 64751 ( Fab4C8-EP54-C5aR2) and 664 PDB ID- 9V4D, EMD- 64777 (Fab4C8-R8Y-C5aR2). 665

666 General chemicals and reagents 667 Most of the general reagents were purchased from Sigma-Aldrich unless otherwise specified. 668 Dulbecco’s Modified Eagle’s Medium (DMEM), Trypsin -EDTA, Fetal Bovine Serum (FBS), 669 Phosphate-Buffered Saline (PBS), Hanks ’ Balanced Salt Solution (HBSS), and Penicillin -670 Streptomycin solution were obtained from Thermo Fisher Scientific. HEK -293T cells (ATCC) 671 were maintained in DMEM (Gibco, Cat. No: 12800 -017) supplemented with 10% (v/v) FBS 672 (Gibco, Cat. No: 10270-106) and 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Cat. 673 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 26 No: 15140122) at 37 °C under 5% CO₂. Sf9 cells were cultured in protein-free media (Gibco, 674 Cat. No: 10902 -088) at 27 °C at 135 rpm. The cDNA coding regions of C3aR, C5aR1 and 675 C5aR2 were cloned into the pcDNA3.1 vector with an HA signal sequence and an N-terminal 676 FLAG-tag followed by a TEV site and the receptor sequence. For expression into Sf9 system, 677 the cDNA of C5aR2 was cloned into pVL1393 vector harbouring N -terminal FLAG -tag, 678 followed by N -terminus of the M4 receptor (residues 2 -23), synthesized by GenScript. The 679 constructs used in the NanoBiT -based β -arrestin recruitment assay were cloned into the 680 pCAGGS vector, with SmBiT fused to the receptor's C -terminus and LgBiT fused to the N -681 terminus of βarr1/2, as previously described 81. The G-protein subunit constructs used in the 682 dissociation assays were generously provided by Asuka Inoue. SRF and SRE reporter gene 683 plasmids were purchased from Promega (Cat. No: E150 for both SRE and SRF). C5aR1 and 684 C5aR2 mutants were generated by using the Q5 ® Site-Directed Mutagenesis Kit (NEB, Cat. 685 No: E0554S). All DNA constructs were verified by sequencing at Macrogen. Peptides, EP54, 686 C5apep, EP67, EP141, P32 and P59 were synthesized by GenScript. Antibodies were 687 purchased from Sigma-Aldrich (M2-HRP coupled anti-FLAG), GenScript (HRP-coupled anti-688 rabbit), or Cell Signaling Technology (ERK1/2), pIMAGO kit purchased from Sigma -Aldrich 689 (Cat No. 18419), hC5aR1 phosphorylation specific antibodies pT324/pS327 and p332/pS334 690 purchased from 7TM antibodies (Cat No. 7TM0032A and 7TM0032B, respectively). 691 Human cell line 692 HEK-293T cells were procured from ATCC and regularly monitored under bright -field 693 microscope for proper morphology, however the examination of mycoplasma contamination 694 was not performed. The cell line was maintained in DMEM supplemented with 10% FBS, 100 695 U/mL penicillin and 100 µg/mL streptomycin, at 37 °C in 5% humidified CO 2 incubator. The 696 cells were maintained at 70-80% confluency either in T175 flasks or 10 cm cell-culture treated 697 round dishes and sub-cultured every alternate day. 698 Chinese hamster ovary (CHO-cells) 699 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 27 Chinese hamster ovary cells stably expressing either C3aR (CHO -C3aR) or C5aR1 (CHO -700 C5aR1) were maintained in Ham’s F12 media supplemented with 10% FCS, 100 U/mL 701 penicillin, 100 µg/mL streptomycin and 400 µg/mL G418 (Invivogen, San Diego, USA). The 702 cell line was maintained in T175 flasks (37 °C, 5% CO 2) and sub-cultured at 90% confluency 703 using TrypLE Express (Thermo Fisher Scientific, Melbourne, Australia). 704 Insect cell line 705 Spodoptera frugiperdA (Sf9) cells were obtained from Expression systems. The cells were 706 maintained in glass conical flasks at a density of 0.9 million cells per mL in protein-free insect 707 cell medium with regular splitting at every alternate day. These cells were grown in a shaker 708 incubator at 27 °C with a constant agitation at 135 rpm. 709 Bacterial cell culture 710 Escherichia coli strain DH5alpha were used for plasmid DNA amplification and isolation, and 711 they were cultured in Luria -Bertani (LB) broth at 37 °C with shaking at 160 rpm. For protein 712 expression, BL21 (DE3), Rosetta (DE3), SHuffle strains of Escherichia coli were used, and 713 they were cultured using Luria -Bertani (LB), Terrific Broth (TB), or 2XYT media under the 714 indicated culture conditions (temperature and shaking) as described in the subsequent 715

sections. 716 Primary Cell culture 717 Human monocyte-derived macrophages (HMDMs) were generated and cultured as previously 718 described82, with experiments approved by The University of Queensland Human Research 719 Ethics Committee. Briefly, human buffy coat blood from anonymous healthy donors was 720 obtained through the Australian Red Cross Blood Service (Brisbane, Australia). Human 721 CD14+ monocytes were isolated from blood using Lymphoprep density centrifugation 722 (STEMCELL, Melbourne, Australia) followed by CD14+ MACS magnetic bead separation 723 (Miltenyi Biotec, Sydney, Australia). The isolated monocytes were differentiated for 7 days in 724 Iscove's Modified Dulbecco's Medium supplemented with 10% FBS, 100 IU/mL penicillin, 100 725 μg/mL streptomycin and 15 ng/mL recombinant human macrophage colony stimulating factor 726 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 28 (BioLegend, San Diego, USA) on 10 mm square dishes (Bio -strategy, Brisbane, Australia). 727 Non-adherent cells were removed by washing with DPBS, and the adherent differentiated 728 HMDMs were harvested by gentle scraping. 729 Mouse bone marrow-derived macrophages (BMDMs) were obtained and cultured as 730 previously described83,84. Briefly, mice were sacrificed by cervical dislocation. The tibia was 731 removed and sterilised. Upon removal of both epiphyses, bone marrow cells were harvested 732 by flushing the central cavity with complete RPMI -1640 medium using a 10 mL syringe 733 attached to a 25 -gauge needle. Cells were then cultured in complete RPMI -1640 medium 734 (containing 10% FBS, 100 IU/mL penicillin, 100 μg/mL streptomycin) supplemented with 735 100 ng/mL recombinant mouse macrophage colony stimulating factor on 10 mm square dishes 736 (Thermo Fisher Scientific, Melbourne, Australia). Mature adherent macrophages for assays 737 were harvested on day 6 by gentle scraping. 738 NanoBiT-based GoA dissociation assay 739 Ligand-induced G-protein activation was measured using a previously described NanoBiT -740 based G-protein dissociation assay85. Briefly, HEK-293T cells were transiently transfected with 741 G-protein subunits harbouring LgBiT -tagged Gα subunit (1 µg), SmBiT -tagged Gγ2 (C68S 742 mutation) subunit (4 µg) and untagged Gβ1 subunit (pcDNA3.1) (4 µg), along with the N -743 terminal FLAG -tagged human (h) and mouse (m) C5aR1 receptor. After 14 -16 h of 744 transfection, the cells were trypsinized and harvested using Trypsin -EDTA and resuspended 745 in NanoBiT buffer (5 mM HEPES, pH 7.4, 1x HBSS, 0.01% BSA, and 10 µM coelenterazine 746 (Gold Bio, Cat. No: CZ5). 100 μL of resuspended cells were then seeded into a 96-well plate 747 at a density of 0.1 million cells per well and incubated at 37 °C for 90 min, followed by an 748 additional 30 min incubation at room temperature. Basal luminescence was recorded for three 749 cycles using a Fluostar Omega plate reader. Subsequently, the cells were stimulated with 750 varying doses of ligand and decrease in luminescence was recorded as a functional readout 751 of G-protein activation for 10 cycles. For analysis, response recorded at 10 min was basal 752 corrected and % decrease in luminescence as a function of ligand concentration was plotted 753 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 29 by normalising luminescence values with respect to the lowest ligand dose taken as 100%. 754 The resulting data was plotted using GraphPad Prism 10.3.1 software, undertaking non-linear 755 regression curve-fitting. 756 GloSensor assay to measure cAMP response 757 To measure ligand -induced change in intracellular cAMP levels, GloSensor assay was 758 employed as previously described86. Briefly, HEK-293T cells were transiently transfected with 759 3.5 μg of either, hC5aR1 or mC5aR1 (cloned in the pcDNA vector having N -terminus FLAG- 760 tag), along with 3.5 μg of the F22 plasmid (Promega, Cat. No: E2301). After 14 –16 h of 761 transfection, cells were trypsinized and resuspended in an assay buffer comprising 20 mM 762 HEPES, pH 7.4, 1x HBSS, and 0.5 mg/mL D -luciferin (GoldBio, Cat. No: LUCNA -1G). 763 Following this, 100 µL of transfected cells were seeded into a 96-well plate at a density of 0.2 764 million cells per well. The cells were incubated at 37 °C for 90 min, followed by an additional 765 30 min incubation at room temperature. Basal luminescence was measured for three cycles. 766 Subsequently, 5 μM forskolin was added to each well, and forskolin induced increase in 767 luminescence was recorded till saturation, i.e., for 8 cycles. Finally, the ligand was added at 768 the indicated doses, and ligand induced decrease in luminescence was recorded for 15 cycles. 769 For data analysis, ligand-mediated decrease in cAMP response was normalised with forskolin-770 induced luminescence and the percentage decrease in cAMP (% normalisation) was 771 calculated by normalising the values of each dose with the smallest ligand dose taken as 772 100%. Curve fitting was done by non -linear regression curve-fitting, using GraphPad Prism 773 10.3.1 software. 774 NanoBiT-based β-arrestin1/2 recruitment 775 To measure agonist induced β-arrestin1/2 recruitment downstream to (h/m) C5aR1 and (h/m) 776 C5aR2, NanoBiT -based enzyme complementation assay was employed, as described 777 previously85. Briefly, HEK-293T cells were transiently transfected with either 3.5 µg hC5aR1/ 778 3.5 µg mC5aR1/ 5 µg hC5aR2/ 0.1 µg mC5aR2, harbouring SmBiT fragment at carboxyl 779 terminus and β-arrestin1/2, 3.5 µg (for h/mC5aR1) / 2 µg (for h/mC5aR2), harbouring LgBiT 780 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 30 fragment at N -terminus using polyethylenimine (PEI) at 1:3 (DNA: PEI) ratio. Post 16 -18 h, 781 cells were harvested using Trypsin -EDTA and resuspended in NanoBiT assay buffer 782 containing 1x HBSS, 5 mM HEPES, pH 7.4, 0.01% BSA and 10 µM coelenterazine. 0.1 million 783 cells were seeded in each well of a 96 well plate and incubated at 37 °C in a 5% humidified 784 CO2 incubator for 90 min. Subsequently, the plates were incubated at room temperature for 785 an additional 30 min, following this the basal luminescence was recorded for 3 cycles using a 786 plate reader (LUMIStar Omega, BMG LABTECH). The cells were then stimulated with varying 787 ligand doses prepared in NanoBiT drug buffer (1x HBSS and 5 mM HEPES, pH 7.4) and 788 increase or decrease in luminescence was recorded for 12 cycles. The average luminescence 789 for 6 cycles (5 -10 cycles) was normalized with respect to luminescence recorded for lowest 790 ligand dose taken as 1. Fold response was plotted as a function of logarithmic dose of ligand 791 using GraphPad Prism 10.3.1 software. Bias factor was calculated by using 792 https://biasedcalculator.shinyapps.io/calc/. 793 Receptor surface expression 794 The surface expression of the receptors was quantified using whole cell-surface ELISA assay, 795 as described previously 87. Briefly, the transiently transfected cells were seeded in poly -D-796 lysine coated 24-well plate at a density of 0.2 million cells per well and incubated at 37 °C in 797 5% humidified CO2 incubator. Post 24 h, cells were washed with 1x Tris -Buffer Saline (TBS) 798 and fixed with 4% paraformaldehyde (PFA) (w/v prepared in 1x TBS) for 20 min followed by 799 washing with 1X TBS, three times, to completely remove the traces of PFA. Subsequently, the 800 cells were incubated with 1% BSA (prepared in 1x TBS) for 1 h followed by incubation in anti-801 FLAG M2-HRP antibody (at 1:10,000 dilution; prepared in 1% BSA) (Sigma, Cat. No. A8592) 802 for another 1 h. After 1 h, the cells were washed three times with 1% BSA to remove excess 803 antibody and ELISA was developed using TMB-ELISA substrate (Thermo Scientific, Cat. No: 804 34028). For the same, the cells were incubated with 200 µL TMB-ELISA substrate until light 805 blue colour appeared. The reaction was quenched by adding 100 µL of above coloured 806 solution into 100 µL 1M H2SO4 and the resultant yellow colour intensity was measured by 807 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 31 reading the absorbance at 450 nm using a multi-mode plate reader (PerkinElmer VictorTM X4). 808 The cell count was quantified using Janus -green stain. For this, the TMB substrate was 809 removed by washing the cells with 1x TBS and incubated with 0.2% (w/v) Janus green B stain 810 (Sigma, Cat. No: 201677) stain for 10 min. The excess stain was removed by washing the 811 cells with MilliQ water. The retained dark -blue colour was dissolved by adding 800 µL of 0.5 812 N HCl and absorbance was recorded at 595 nm. To quantify the receptor surface expression, 813 the ratio of absorbance at 450 nm and 595 nm was taken and normalized with respect to the 814 mock (pcDNA) transfected cells and plotted using GraphPad Prism 10.3.1 software. 815 Intracellular calcium mobilisation assays 816 Ligand-induced intracellular calcium mobilisation was assessed using Fluo -4 NW Calcium 817 Assay kit following the manufacturer’s instructions (Thermo Fisher Scientific, Melbourne, 818 Australia), as described previously11. Briefly, HMDMs (70,000 per well) or BMDMs (90,000 per 819 well) were seeded in black clear -bottom 96-well tissue culture plates overnight. Cells were 820 firstly stained with the Fluo -4 dye in assay buffer (1x HBSS, 20 mM HEPES) for 50 min 821 (37 °C, 5% CO2). C5a/C5a-d-Arg dilutions were prepared in assay buffer containing 0.1% BSA. 822 On a Flexstation 3 platform, the fluorescence (Ex/Em: 494/516 nm) was continually monitored 823 for a total of 100 s, with ligand addition performed at 16 s. Data were recorded as the 824 magnitude of signal deviation from the baseline. 825 Measuring ERK and RhoA signaling 826 For measuring ERK signaling downstream to stimulation of hC5aR1 and mC5aR1 with 827 ligands, we undertook an SRE reporter assay88. HEK-293T cells were transfected with 3.5 μg 828 of N -terminally FLAG -tagged hC5aR1/mC5aR1 and 3.5 μg of an SRE -based luciferase 829 reporter plasmid pGL4.33 (Promega, Cat. no: E1340). 14 -16 h post-transfection, cells were 830 washed with 1x PBS, trypsinized and seeded into a 96-well plate at a density of 1 x 10 6 cells 831 per well in the presence of complete media. Cells were allowed to settle for 8 h, followed by 832 starvation in serum -deprived DMEM (without FBS), for 12 h. Subsequently, the cells were 833 stimulated with the indicated dose of the ligands (prepared in serum -free DMEM) and the 834 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 32 plates were incubated at 37 °C for 3 h. Prior to reading, the serum -free media was carefully 835 replaced with the assay buffer containing 20 mM HEPES pH 7.4 and 1x HBSS supplemented 836 with 0.5 mg/mL D -luciferin. Luminescence was recorded immediately. Signal observed was 837 normalized with respect to the luminescence observed at lowest concentration of each ligand, 838 taken as 1. Data was plotted and analyzed using GraphPad Prism 10.3.1 software. 839 For measuring RhoA signaling, HEK -293T cells were transfected with 3.5 μg of N -840 terminally FLAG-tagged receptor and 3.5 μg of an SRF -based luciferase reporter plasmid 841 pGL4.34 (Promega, Cat. no: E135A) followed by the same steps as that in SRE reporter assay. 842 The ligand -induced ERK1/2 phosphorylation in HMDM and BMDM was assessed 843 using the AlphaLISA Surefire Ultra p-ERK1/2 (Thr202/Tyr204) kit (Revvity, Melbourne, 844 Australia) as previously described 89. Briefly, HMDMs (50,000/well) or BMDMs (90,000/well) 845 were seeded in tissue culture -treated 96-well plate for 24 h and serum -starved overnight. 846 Human and mouse (h/m) C5a/C5a-d-Arg dilutions were prepared in serum -free medium 847 containing 0.1% BSA and added to the cells (10 min for HMDMs; 5 min for BMDMs). After 848 stimulation, cells were immediately lysed using AlphaLISA lysis buffer on a microplate shaker 849 (450 rpm, 10 min). For the detection of phospho -ERK1/2 content, cell lysate ( 5 μL/well) was 850 transferred to a 384-well ProxiPlate (Revvity) and added to the donor and acceptor reaction 851 mix (2.5 μL/well, respectively) with 2 h incubation at room temperature in the dark. The plate 852 was read on Tecan Spark 20M following standard AlphaLISA settings. 853 For measuring agonist -induced ERK1/2 phosphorylation in C5aR1 -expressing HEK-854 293 stable cell line, a previously described Western blotting–based protocol was employed90. 855 Briefly, hC5aR1 expressing stable cell lines were seeded into a 6 -well plate at a density of 1 856 million cells per well. The cells were serum-starved for 12 h followed by stimulation with 1 μM 857 concentration of hC5a and hC5a-d-Arg, at selected time points. After the stimulation, the medium 858 was aspirated, and the cells were lysed in 100 μL of 2x SDS dye per well. The cells were 859 heated at 95 °C for 15 min, followed by centrifugation at 15,000 rpm for 15 min. 10 μL of lysate 860 was loaded per well and separated on SDS -PAGE, followed by Western blotting. The blots 861 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 33 were blocked in 5% BSA (in 1x TBST) for 1 h and incubated overnight with rabbit -raised 862 phospho-ERK (Cat no. 9101/CST) primary antibody at 1:5000 dilution. Following this, the blots 863 were washed thrice with 1x TBST for 10 min each and incubated with anti-rabbit HRP-coupled 864 secondary antibody (1:10,000, Cat no. A00098/Gen Script) for 1 h. The blots were washed 865 again with 1x TBST three times and developed with Promega ECL solution (Cat. No: W1015) 866 using ChemiDoc (Bio-Rad). The blots were stripped with a low pH stripping buffer and then 867 re-probed for total ERK using rabbit -raised p44/42 MAPK (Erk1/2) Antibody (Cell Signalling 868 Technology, Cat. No: 9102) primary antibody at 1:5000 dilution. Blots were again incubated 869 with anti-rabbit HRP-coupled secondary antibod y for 1 h after washing three times with 1x 870 TBST buffer. ERK phosphorylation signal was obtained after quantification with ImageJ 871 software and fold normalized values were plotted using GraphPad Prism 10.3.1 software. 872 For measuring ligand pERK1/2 signaling in response to BM2020 -7 and BM2020 -8 873 analogues, ligand-induced phospho-ERK 1/2 signaling was assessed using the Alpha LISA 874 SureFire Ultra pERK 1/2 (Thr202/Tyr204) assay kit (PerkinElmer, Melbourne, Australia). 875 Either CHO -C3aR or CHO -C5aR1 cells were seeded (50,000 cells per well) onto 96 -well 876 tissue culture-treated plates, incubated for 24 h and subsequently serum starved overnight. 877 Ligand dilutions were prepared in serum-free medium. Cells were stimulated with respective 878 ligands for 10 min and then immediately lysed using AlphaLISA lysis buffer. Cell lysate (5 µL 879 per well) was added to a 384-well ProxiPlate (PerkinElmer, Melbourne, Australia) followed by 880 the donor and acceptor reaction mixes (2.5 µL per well each). Following a 2 h incubation in 881 the dark, the plate was read on a CLARIOstar Plus microplate reader following standard 882 AlphaLISA settings. Experiments were conducted in triplicate and conducted on at least three 883 different days. Data were analysed using GraphPad software (Prizm 10.1) and expressed as 884 mean ± standard error of mean (SEM). For each repeat, data was normalised prior to being 885 combined. Logarithmic concentration-response curves were plotted using combined data and 886 analysed to calculate the potencies of each peptide. 887 Measurement of IL8 release using ELISA 888 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 34 To compare the ability of the C5a species to induce IL8 release from HMDMs82, HMDMs were 889 seeded in 96-well tissue culture plates (90,000 per well) for 24 h before treatment. Cells were 890 stimulated with various concentrations of human plasma -derived or recombinant C5a/ 891 C5a-d-Arg for 24 h (37 °C, 5% CO 2) before the supernatant was collected. The IL8 level in the 892 supernatant was quantified using human IL8 enzyme-linked immunosorbent assay (ELISA) kit 893 (BD OptEIA) as per manufacturer’s protocol. 894 PMN chemotaxis assay using FluroBlok 895 C5a-induced chemotaxis of human polymorphonuclear leukocytes (PMNs) were assessed 896 using Corning® FluoroBlok™ HTS 96-well Multiwell Permeable Support System (Corning, New 897 York, USA). PMNs were obtained from venous whole blood (20 mL) collected from healthy 898 volunteers under informed consent. Samples were collected using venepuncture into BD 899 K2EDTA Vacutainer® blood collection tubes (BD Biosciences, Macquarie Park, Australia) and 900 processed within 5 h. For PMN isolation, the anticoagulated blood was firstly layered over a 901 Lymphoprep (STEMCELL, Melbourne, Australia) density gradient and then centrifuged 902 (800xg, 30 min, 22 °C), followed by residual erythrocytes removal using hypotonic lysis 91. 903 Isolated PMNs were counted and resuspended in HBSS, 20 mM HEPES, 0.5% BSA migration 904 buffer (2 x 106 per mL). Calcein AM (2 µM) was added to label the cells (30 min, 37 °C). The 905 cells were then gently washed once with HBSS buffer and added to the insert (2 x 10 5 per 906 insert). To initiate cell migration, C5a/C5a-d-Arg prepared in the migration buffer were added to 907 the receiver wells. On a Tecan Spark 20M microplate reader (Tecan, Männedorf, Switzerland) 908 (37 °C), ligand-induced cell migration was continuously monitored at 2 min intervals for 40 min 909 by quantifying Calcein AM fluorescence from the receiver -side of the insert (Ex/Em = 485 910 nm/525 nm). The relative cell migration (fold-baseline) at 20 min post ligand addition was used 911 for graphing. 912 BRET assay measuring β-arrestin recruitment to C5aR1 913 The C5a-mediated β-arrestin recruitment to C5aR1 was measured using bioluminescence 914 resonance energy transfer (BRET) -based assay using methods described elsewhere 83,89. 915 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 35 Briefly, HEK-293 cells were transiently transfected with human C5aR1 -Rluc8 and β-arrestin 916 1/2-venus constructs using XTG9 (Merck, Melbourne, Australia) for 24 h. Transfected cells 917 were then seeded (100,000 per well) onto white 96 -well plates (Corning, New York, USA) in 918 phenol-red free DMEM containing 5% FBS overnight. For BRET assay, cells were first 919 incubated with the substrate Enduren (30 µM, Promega, Sydney, Australia) for 2 h (37 °C, 5% 920 CO2). On a Tecan Spark 20M microplate reader (Tecan, Männedorf, Switzerland) maintained 921 at 37 °C, the BRET light emissions (460 -485 and 520-545 nm) were continuously monitored 922 for 90 min with C5a/C5a -d-Arg added after 15 min. The ligand -induced BRET ratio was 923 calculated by subtracting the emission ratio of Venus (520-545 nm)/Rluc8 (460-485 nm) of the 924 vehicle-treated wells from that of the ligand -treated wells. The data at 40 min post ligand 925 addition was used for derivation of concentration-response curves. 926 For measuring β -arrestin 2 recruitment in response to BM2020 -7 and BM2020 -8 927 analogous peptides, HEK -293 WT cells were transiently transfected with C5aR1 -Renilla 928 Luciferase 8 (Rluc8) and β-arrestin 2-Venus or C5aR2-Venus and β-arrestin 2-Rluc constructs 929 using X-tremeGENE 9 DNA Transfection Reagent (Roche, Sydney, Australia). The following 930 day, adherent cells were detached using TrypLE Express and seeded plates (100,000 cells 931 per well) onto white 96 -well tissue culture -treated plates (Corning, NY, USA) in phenol -red 932 free DMEM supplemented with 5% FBS. On the next day, the cells were incubated with either 933 Enduren (C5aR1, Promega, Sydney, Australia) or Endurazine (C5aR2, Promega) substrates 934 diluted in assay media for 2 h (37 °C, 5% CO 2). BRET emissions (460-485 nm and 520-545 935 nm) were measured using either a Tecan Spark 20M or PHERAstar FSX microplate reader 936 (37 °C) for 19 reads, with respective ligands added after the first 4 reads. The ligand induced 937 BRET ratio was calculated by subtracting the Venus/Rluc8 ratio of the negative control wells 938 from that of the ligand treated wells. 939 Neutrophil mobilisation in mouse 940 Eight- to ten-week-old C57BL/6J mice (n=4 per group; Ozgene, Australia) received a single 941 intraperitoneal dose of the C5aR1 receptor antagonist PMX205 (3 mg/kg) using a 942 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 36 concentration of 1.5 mg/mL in 5% (w/v) dextrose (Baxter, cat. no. AHB0063) or an equal 943 volume of vehicle (5% (w/v) dextrose). Fifteen minutes later, recombinant mC5a or mC5a-d-Arg 944 (produced in-house) diluted in sterile 0.9% (w/v) sodium chloride for injection (Pfizer, Cat. no. 945 1016696) was administered intravenously via the lateral tail vein at 50 µg/kg. Peripheral blood 946 (20 µL) was collected from the tail tip of mice at 0 min and at 60 min after C5a challenge, then 947 mixed with 480 µL V -52D diluent (Mindray, Cat. no. 105 -005962-00). Absolute neutrophil 948 counts were measured using an automated haematology analyser (BC -5000; Mindray, 949 Shenzhen, China). All animal procedures were approved by the University of Queensland 950 Animal Ethics Committee and performed in accordance with ARRIVE guidelines. 951 MD simulation 952 Receptor dynamics-driven important functional features of GPCRs 47,48 have been simulated 953 for the C5aR1 using the AceMD engine92. Receptors (PDB code: 8IA2, 8JZZ) were prepared 954 in MOE software (www.chemcomp.com). Systems were generated in Charmm-GUI software93 955 with parameters from the CharMM36M forcefield94. Each mutated system was generated with 956 Charmm-GUI93, using the structure of the C5a -bound human C5aR1 (PDB code: 8IA2) as a 957 starting point. The complexes were solvated (TIP3P water) and neutralized using a 0.15 958 concentration of NaCl ions. All systems underwent 100 ns equilibration in conditions of 959 constant pressure (NPT ensemble, pressure maintained with Berendsen barostat, 960 1.01325 bar pressure), using a timestep of 2 fs. During this stage restraints were applied to 961 the protein and ligand backbone. This was followed by 3 separate NVT runs for each system, 962 1 µs each. For each of the simulations we used a temperature of 310 K, which was maintained 963 using the Langevin thermostat, hydrogen bonds were restrained using the RATTLE algorithm. 964 Non-bonded interactions were cut -off at a distance of 9 Å, with a smooth switching function 965 applied at 7.5 Å. The simulation data have been uploaded to the GPCRmd repository 48: 966 https://gpcrmd.org/dynadb/publications/XXXX/. 967 Hydrogen-Deuterium exchange-mass spectrometry (HDX-MS) 968 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 37 mC5aR1 and C5aR2 samples were prepared at 100 µM in 20 mM HEPES pH 7.4, 150 mM 969 NaCl, and 0.01% L-MNG. The protein samples (3.4 µL) were mixed with 26.6 µL D2O buffer 970 (20 mM HEPES pH 7.4, 150 mM NaCl, and 10% glycerol), incubated for 10, 100, 1000, and 971 10000 s at room temperature (23-25 °C), and quenched with 30 µL of ice-cold quench buffer 972 (60 mM NaH2PO4 pH2.01, 20 mM TCEP, 2 M GuHCl). The quenched samples were then 973 snap-frozen on dry ice and stored at -80 °C. For non -deuterated samples, 3.4 µL of protein 974 samples were mixed with 26.6 µL of their respective H 2O buffers and quenched with the 30 975 µL quench buffer. 976 The quenched samples were thawed and immediately digested by passing through an 977 immobilized pepsin column (2.1 x 30 mM) (Life Technologies, Carlsbad, CA, USA) at a flow 978 rate of 100 µL/min in 0.05% formic acid in H2O at 12 °C. The peptic fragments were collected 979 on a C18 VanGuard trap column (1.7 µM x 30 mM) (Waters) for desalting with 0.05% formic 980 acid in H2O. The peptic fragments were then separated by an Acuity UPLC C18 column (1.7 981 µm, 1.0 x 100 mM) (Waters) at a flow rate of 40 µL/min in mobile phase A (0.1% formic acid 982 in H2O) with an acetonitrile gradient increase starting from 8% to 85% over 8.5 min with mobile 983 phase B (0.1% formic acid in acetonitrile). To minimize the back -exchange, the buffers were 984 adjusted to pH 2.5, and the analysis was performed at 0.5 °C. Mass spectral analyses were 985 performed using a Xevo G2 quadrupole time-of-flight (Q-TOF) equipped with a standard ESI 986 source in MSE mode (Waters) in positive ion mode. The capillary, cone, and extraction cone 987 voltages were set to 3 kV, 40 V, and 4 V, respectively. The source and desolvation 988 temperatures were set at 120 °C and 350 °C, respectively. Trap and transfer collision energies 989 were set to 6 V; the trap gas flow was 0.3 m L/min. Before analysis, the mass spectrometer 990 was calibrated by sodium iodide (2 µg/µL). [Glu1]-Fibrinopeptide B (200 fg/µL) in MeOH:water 991 (50:50 (v/v) + 1% acetic acid) was utilized for lock -mass correction. The ions at mass -to-992 charge ratio (m/z) of 785.8427 were monitored at a scan time of 0.1 s with a mass window of 993 ± 0.5 Da. The reference internal calibrant was introduced into the lock-mass sprayer at a flow 994 rate of 20 µL/min, and all spectra were automatically corrected. Two independent interleaved 995 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 38 acquisition functions were created: the first function, typically set at 4eV, collected low-energy 996 or unfragmented data, and the second function collected high -energy or fragmented data 997 typically obtained using a collision ramp from 30 -55 eV. Mass spectra were acquired in the 998 range of m/z 100-2000 for 10 min. The peptides from non-deuterated samples were identified 999 with ProteinLynx Global Server 2.4 (Waters), and the level of deuterium uptake for each 1000 peptide was determined by measuring the centroid of the isotopic distribution with DynamX 1001 3.0 (Waters). Back -exchange was not corrected because we observed protein aggregates 1002 when preparing the fully deuterated samples. 1003 NanoBiT-based GRK recruitment assay 1004 Ligand-induced GRK recruitment to C5aR1 was measured by a NanoBiT -GRK assay85 with 1005 minor modifications. Specifically, HEK-293A cells (Thermo Fisher Scientific) were seeded in 1006 a 6-well culture plate at a density of 0.2 million cells per mL (2 mL per well in DMEM (Nissui) 1007 supplemented with 5% FBS, glutamine, penicillin, and streptomycin) one day before 1008 transfection. The transfection solution was prepared by mixing 5 µL (per well) of 1009 polyethyleneimine (PEI) Max solution (1 mg/mL; Polysciences), 200 µL of Opti-MEM (Thermo 1010 Fisher Scientific), and a plasmid mixture containing 500 ng of sHA -FLAG-C5aR1-LgBiT and 1011 500 ng of GRK -SmBiT. After a day of incubation, the transfected cells were harvested with 1012 Dulbecco's PBS containing 0.5 mM EDTA, centrifuged, and resuspended in 3 ml of HBSS 1013 containing 0.01% bovine serum albumin (BSA; fatty acid -free grade; SERVA) and 5 mM 1014 HEPES, pH 7.4 (assay buffer). The cell suspension was dispensed into a white 96-well plate 1015 at a volume of 80 µL per well, and 20 µL of 50 µM coelenterazine (Angene) diluted in the 1016 assay buffer was added. After a 2 h incubation at room temperature, baseline luminescence 1017 was measured using a SpectraMax L (Molecular Devices), and a titrated test ligand (20 µL; 1018 6x of final concentrations) was manually added. The plate was immediately read at room 1019 temperature in kinetics mode for 15 min, with measurements taken every 20 s. Luminescence 1020 counts from 5–10 min after ligand addition were averaged and normalized to the initial counts. 1021 The fold-change values were further normalized to those of vehicle-treated samples and used 1022 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 39 to plot the GRK recruitment response. GRK recruitment signals were fitted to a four-parameter 1023 sigmoidal concentration-response curve using Prism 10 software (GraphPad Prism 10.3.1 1024 software). For each replicate experiment, the parameter Span (= Top – Bottom) for individual 1025 ligands was normalized to acetylcholine, and the resulting Emax values were used as a 1026 measure of efficacy. 1027 Receptor phosphorylation measurement via pIMAGO assay 1028 Agonist-induced phosphorylation downstream of mC5aR1 was measured by using pIMAGO 1029 phosphoprotein detection kit from Sigma (Cat. No. 18419) , following the manufacturer’s 1030 protocol. Briefly, Sf9 cells were co-infected with baculovirus expressing mC5aR1 and either 1031 GRK2 or GRK 6 at a density of 1.8 million cells per mL. 72 h post -infection, cells were 1032 stimulated with either 1 µM mC5a or mC5a-d-Arg for 30 min at 37 °C. Following stimulation, cells 1033 were harvested by centrifugation at 5,000 rpm for 10 min. The harvested pellets were 1034 processed for lysis, and cells were dounce -homogenized in lysis buffer containing 20 mM 1035 HEPES, pH 7.4, 150 mM NaCl, 1x PhosSTOP (Roche, Cat. No. 57084100), and 1x protease 1036 inhibitor cocktail (Roche, Cat. No. 04693116001). Lysates were solubilized in 1% (w/v) L-MNG 1037 (Cat. No. NG31025GM) at room temperature for 1 h and centrifuged at 15,000 rpm for 10 min. 1038 The cleared lysate was transferred to a separate tube containing pre -equilibrated M1-FLAG 1039 beads supplemented with 5 mM CaCl₂. Samples were incubated at room temperature for 90 1040 min with gentle tumbling to allow bead binding. The beads were washed five times with low-1041 salt buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM CaCl₂, and 0.01% L-MNG) alternated 1042 with high-salt buffer (20 mM HEPES, pH 7.4, 350 mM NaCl, 2 mM CaCl₂, and 0.01% L-MNG). 1043 Bound proteins were eluted using FLAG-elution buffer containing 20 mM HEPES, pH 7.4, 150 1044 mM NaCl, 2 mM EDTA, 0.01% L-MNG, and 250 µg/mL FLAG peptide. Subsequently, protein 1045 loading dye was added to each sample, followed by the addition of 5x IAA solution to a final 1046 1x concentration from the pIMAGO kit. The samples were incubated at room temperature for 1047 15 min in the dark. After incubation, samples were subjected to SDS -PAGE, followed by 1048 western blotting. The PVDF membrane was blocked in 1x blocking buffer overnight, then 1049 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 40 incubated with the pIMAGO reagent (1:1000, prepared in 1x pIMAGO buffer) for 1 h. The 1050 membrane was washed three times with 1x wash buffer (prepared from 10x stock) and once 1051 with 1x TBST (for 5 min each). The PVDF membrane was then incubated with avidin -HRP 1052 (1:1000, prepared in 1x blocking buffer) for 1 h at room temperature, followed by three washes 1053 with 1x TBST (5 min each). The signal was detected using the Promega ECL solution on a 1054 ChemiDoc imaging system (Bio-Rad). The PVDF membrane was subsequently stripped and 1055 re-probed for total receptor levels using HRP -conjugated anti -FLAG M2 antibody (Sigma, 1056 1:5000). Phosphorylation and receptor signals were quantified using ImageLab software (Bio-1057 Rad). Fold-normalized values were plotted using GraphPad Prism 10.3.1 software. 1058 Detection of phosphorylation status of receptor via hC5aR1 specific antibodies 1059 To detect ligand -induced phosphorylation downstream of hC5aR1, HEK293 -T cells were 1060 transfected with 7 µg of hC5aR1 using the PEI transfection reagent. 48 h post -transfection, 1061 cells were starved for 6 h in serum -free DMEM. Following starvation, cells were stimulated 1062 with 1 µM of hC5a or hC5a -d-Arg for 30 min at 37 °C. After stimulation, cells were scraped, 1063 collected, and harvested by centrifugation at 10,000 rpm for 10 min. The resulting pellet was 1064 dounce-homogenized in lysis buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl, 1x 1065 PhosSTOP (Roche, Cat. No. 57084100), and 1x protease inhibitor cocktail (Roche, Cat. No. 1066 04693116001). Lysates were then solubilized in 1% (w/v) L-MNG at room temperature for 90 1067 min and centrifuged at 15,000 rpm for 10 min. The cleared lysate was transferred to a separate 1068 tube containing pre-equilibrated M1-FLAG beads supplemented with 5 mM CaCl ₂. Samples 1069 were tumbled at room temperature for 90 min to allow bead binding. The beads were then 1070 washed five times, alternating between low-salt buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1071 2 mM CaCl₂, and 0.01% L-MNG) and high-salt buffer (20 mM HEPES, pH 7.4, 350 mM NaCl, 1072 2 mM CaCl ₂, and 0.01% L -MNG). Bound proteins were eluted using FLAG -elution buffer 1073 containing 20 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM EDTA, 0.01% L-MNG, and 250 µg/mL 1074 FLAG peptide. After elution, 5x reducing dye was added to the sample, and proteins were 1075 separated by SDS -PAGE for western blotting. The PVDF membrane was blocked with 5% 1076 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 41 BSA prepared in 1x TBST for 1 h at room temperature. The membrane was then incubated 1077 with primary antibodies (1:5000 dilution) specific for p324/327 and p332/334 for 1 h at room 1078 temperature. Following incubation, the membrane was washed three times with 1x TBST and 1079 incubated with an anti-rabbit HRP-conjugated secondary antibody for 1 h at room temperature. 1080 This was followed by three additional washes with 1x TBST. The signal was detected using 1081 the Promega ECL solution on a ChemiDoc imaging system (Bio -Rad). The membrane was 1082 subsequently stripped and re-probed for total receptor expression using an HRP -conjugated 1083 anti-FLAG M2 antibody (Sigma, 1:5000). The phosphorylation signal was quantified using 1084 ImageLab software (Bio -Rad) and normalized to the total receptor signal. Fold -normalized 1085 values were plotted using GraphPad Prism 10.3.1 software. 1086 Purification of h/mC5a, h/mC5a-d-Arg, Trx-hC5a and Trx-hC5a-d-Arg 1087 C5a and C5a -d-Arg from human and mouse were cloned into pET -32a(+) vector with an N -1088 terminus 6x -His-Trx tag followed by TEV -site, and purified using a previously described 1089 protocol50,82, with slight modifications. Briefly, starter culture inoculated with freshly 1090 transformed E. coli SHuffle cells in 50 mL LB media supplemented with 100 μg/mL ampicillin 1091 was grown overnight at 30 °C. This was inoculated in 1 L LB media, similarly, supplemented 1092 with 100 μg/mL ampicillin, and the culture grown at 30 °C till O.D 600 reached 0.8 -1. 1093 Subsequently, the protein expression was induced with 1 mM IPTG and shifted to 16 °C for 1094 overnight induction. Post-harvesting, cells were incubated with 1 mg/mL lysozyme in 50 mM 1095 HEPES, pH 7.4, 300 mM NaCl, 30 mM Imidazole, 1 mM PMSF, and 2 mM benzamidine for 1096 40 min at 4 °C followed by disruption with ultrasonication and removal of cell debris with high-1097 speed centrifugation. Trx-C5a/C5a-d-Arg in the lysate was captured on an Ni-IDA resin (Takara, 1098 Cat. No: 635662) using gravity flow columns. Resin containing bound protein was thoroughly 1099 washed with 50 mM HEPES, pH 7.4, 1 M NaCl, 30 mM Imidazole to remove non -specific 1100 proteins, and Trx-fused-C5a/C5a-d-Arg was eluted with 50 mM HEPES, pH 7.4, 150 mM NaCl, 1101 300 mM Imidazole. To remove imidazole from the eluted protein, it was dialysed overnight in 1102 30 mM HEPES and 150 mM NaCl in 4 °C and stored in 10% glycerol at -80 °C for further use 1103 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 42 in structural determination. To prepare His-Trx-free C5a/C5a-d-Arg, Trx-His tag was cleaved by 1104 incubation for 16 h, at room temperature, with TEV-protease (1:20 w/w, TEV: fusion protein). 1105 Cleaved C5a/C5a-d-Arg, was further separated by cation-exchange chromatography and stored 1106 at -80 °C with 10% glycerol concentration. This was used for functional assays and complexing 1107 unless specified otherwise. 1108 Receptor purification from Sf9 cells 1109 Human and mouse C5aR2 was purified from Sf9 cells following a previously described 1110 protocol12. Briefly, human and mouse C5aR2 expressing baculovirus were employed to set up 1111 infection in Sf9 cells at a density of 2 x 106 cells per mL and allowed to grow for 72 h at 27 °C 1112 followed by harvesting the cells by centrifugation at 5,000 rpm for 15 min. The harvested pellet 1113 was immediately flash frozen in liquid nitrogen and stored at -80 °C until further use. 1114 For purification, the receptor -expressing pellets were thawed and sequentially 1115 homogenised in hypotonic buffer (20 mM HEPES, pH 7.4, 1 mM MgCl 2, 2 mM KCl, 1 mM 1116 PMSF, 2 mM benzamidine) followed by hypertonic buffer (20 mM HEPES, pH 7.4, 1M NaCl, 1117 1 mM MgCl2, 2 mM KCl, 1 mM PMSF, 2 mM benzamidine) and centrifuged at 20,000 rpm for 1118 20 min at 4 °C to remove cytosolic contaminants. Membrane solubilisation was carried out by 1119 resuspending the pellet in solubilisation buffer (20 mM HEPES, pH 7.4, 450 mM NaCl, 1% L-1120 MNG (Anatrace, Cat. no: NG310), 0.1% CHS (Sigma, Cat. no: C6512), 1 mM PMSF, 2 mM 1121 benzamidine) and incubating the lysate for 2 h at 4 °C in the presence of 2 mM iodoacetamide. 1122 Following solubilisation, the lysate was 3-fold diluted in dilution buffer (20 mM HEPES, pH 7.4, 1123 1 mM PMSF, 2 mM benzamidine, 2.5 mM CaCl 2) and subjected to high-speed centrifugation 1124 at 20,000 rpm for 20 min. The resulting supernatant was filtered through 0.45 µ bottle -top 1125 filters (Merck Millipore, Cat. No: HVLP04700) and loaded on to pre -equilibrated gravity flow 1126 columns containing M1 anti-Flag resin (prepared in-house). The unbound and non-specifically 1127 bound proteins were removed by three washes of low salt buffer (20 mM HEPES, pH 7.4, 150 1128 mM NaCl, 2 mM CaCl 2, 0.1% L-MNG, 0.01% CHS) alternated with two washes of high salt 1129 buffer (20 mM HEPES, pH 7.4, 2 mM CaCl 2, 350 mM NaCl, 0.1% L-MNG). The receptor was 1130 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 43 eluted in elution buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 0.01% L -MNG, 2 mM EDTA, 1131 250 µg/ml FLAG-peptide). Finally, free cysteine residues in the receptor were blocked with 2 1132 mM iodoacetamide and excess iodoacetamide was quenched with 2 mM L -cysteine. The 1133 purified receptor was stored in 10% glycerol at -80 °C until further use. 1134 For Apo-C5aR2, the receptor purification was carried out in the absence of any ligand, 1135 however for ligand -bound receptor (Trx -hC5a/Trx-hC5a-d-Arg-C5aR2, mC5a-d-Arg-mC5aR2, 1136 EP54-C5aR2, C5apep-C5aR2, R8Y-C5aR2), either 100 nM Trx-hC5a/hC5a-d-Arg, mC5a-d-Arg or 1137 1 µM peptide ligands were added at each purification step while keeping 1 µM Trx-hC5a/hC5a-1138 d-Arg and mC5a-d-Arg and 10 µM peptide ligands at elution. 1139 Complexing of Trx-hC5a/Trx-hC5a-d-Arg-bound C5aR2 and mC5a-d-Arg-mC5aR2 1140 For the structure determination of Trx -hC5a/Trx-hC5a-d-Arg-bound C5aR2 and mC5a -d-Arg-1141 bound mC5aR2, SEC of the purified ligand-bound receptor was performed with running buffer 1142 having 20 mM HEPES, pH 7.4, 150 mM NaCl, 0.01% L -MNG, 0.001% CHS, supplemented 1143 with 100 nM Trx -hC5a/Trx-hC5a-d-Arg/mC5a-d-Arg, as applicable. The Trx -hC5a/Trx-hC5a-d-Arg-1144 hC5aR2 and mC5a -d-Arg-mC5aR2 were separated on Superose TM 6 Increase 10/300 GL 1145 (Cytiva, Cat. No: 29091596) and Superdex TM 200 Increase 10/300 GL (Cytiva, Cat. No: 1146 28990944), respectively. The elution fractions corresponding to Trx -hC5a/hC5a-d-Arg-bound 1147 C5aR2 were pooled and concentrated to 6 -12 mg/mL using a 100 MWCO concentrator 1148 (Cytiva, Cat. no: 28932319) for Cryo-EM grid preparation. 1149 Cryo-EM grid preparation and data collection 1150 3 μl of the purified Trx-hC5a/hC5a-d-Arg-hC5aR2, mC5a -d-Arg-mC5aR2, and Fab4C8-C5aR2 1151 complexes were applied onto glow discharged Quantifoil holey carbon grids (R1.2/1.3, Au, 1152 300 mesh) at a concentration ranging from 5-20 mg/ml. The grids were blotted for 4 s at 4 °C 1153 and 100% humidity with a blot force of 10 using a Vitrobot Mark IV (Thermo Fischer Scientific) 1154 and immediately plunge frozen in liquid ethane (-181 °C). 1155 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 44 Data collection of all samples was performed on a Titan Krios G3i/G4 (Thermo Fisher 1156 Scientific) operating at an accelerating voltage of 300 kV equipped with a Gatan K3 direct 1157 electron detector and BioQuantum K3 imaging filter. Movie stacks were acquired in counting 1158 mode at a pixel size of 0.83 Å/px and a dosage rate of approximately 7.7 - 8.6 e-/Å2/s using 1159 EPU software over a defocus range of -0.8 to -1.6 μm. Each movie was fractionated into 64 1160 frames with a total dose of 62.1-64.3 e-/Å2 that was obtained throughout the 5.0-6.0 s exposure 1161 period. In total 4,139, 3,200, 3,350, 3,381, 3,576, 4,189 and 1,652 movie stacks were acquired 1162 for Trx-C5a-C5aR2, Trx-C5ad-Arg-C5aR2, mC5a-d-Arg-mC5aR2, Fab4C8-Apo-C5aR2, Fab4C8-1163 C5apep-C5aR2, Fab4C8-EP54-C5aR2 and Fab4C8-R8Y-C5aR2, respectively. 1164 Image processing and map construction 1165 All datasets were processed following a similar pipeline in sub-programs of CryoSPARC72 1166 v4.6, unless stated otherwise. Briefly, dose-fractionated movie stacks were aligned with Patch 1167 Motion Correction (multi), and contrast transfer function (CTF) parameters were estimated 1168 using Patch CTF (multi). Particles were auto-picked using the blob-picker module, extracted 1169 using a box-size of 320 px (Fourier cropped to 64 px) and cleaned using reference -free 2D 1170 classification to remove ice contamination and distorted particles. Selected particles were 1171 subjected to reference free ab-initio reconstruction into multiple classes followed by 1172 heterogenous refinement. The particles corresponding to the best class were re -extracted 1173 using a box size of 320 px (Fourier cropped to 256 px) and subjected to non -uniform (NU) 1174 refinement and local refinement providing reconstructions with resolutions ranging from 2.97 1175 Å – 3.82 Å at Fourier Shell Correlation of 0.143. Local resolutions were estimated using the 1176 LocRes module in cryoSPARC, providing half-maps as input. Details and number of particles 1177 in each step in the processing pipelines of individual complexes are presented in Figure S4 -1178 S5. 1179 Model building and refinement 1180 The coordinates of C5aR2 obtained from Alpha Fold 95 (AF-Q9P296) were used to dock into 1181 the EM maps of Trx -C5a-C5aR2, Fab4C8-Apo-C5aR2, Fab4C8-C5apep-C5aR2, Fab4C8-1182 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 45 EP54-C5aR2 and Fab4C8-R8Y-C5aR2 using UCSF Chimera96. The resulting coordinates and 1183 maps were imported and subjected to “All -atom Refine” in COOT 97-99, followed by iterative 1184 rounds of manual adjustment in COOT and real -space refinement in Phenix 100,101. The final 1185 refined models displayed good geometry, and the refinement statistics for all models are 1186 presented in Table S2. Since the Fab 4C8 was derived from a hybridoma clone and we did 1187 not have the sequence information for the Fab component, we were unable to build models 1188 for the corresponding Fab densities in the map. 1189 Protein-protein contacts and volumes of pockets were calculated using the PDBsum 1190 server102. All figures were prepared using UCSF Chimera or UCSF ChimeraX96,103. 1191 BM2020-7 and BM2020-8 peptide synthesis 1192 Peptide synthesis was performed manually using Fmoc (9 - fluorenylmethyloxycarbonyl)-1193 based solid phase peptide synthesis (SPPS) on 2-chlorotrityl chloride (2-CTC) and Rink Amide 1194 AM resins. A ratio of 4 eq. of amino acid, 4 eq. of HBTU and 8 eq. of DIPEA was used for 1195 each coupling. N-terminal acetylation was performed on -resin using 2 x 5 min treatments of 1196 5% acetic anhydride (Sigma-Aldrich) and 3% DIPEA (Sigma-Aldrich) in DMF (25O C) Following 1197 synthesis, the peptide was cleaved from the resin and side chain protecting groups removed 1198 with a treatment of trifluoroacetic acid (TFA)/triisopropylsilane(TIPS)/water (95:2.5:2.5). 1199 Following cleavage, purification was performed using reverse phase high performance liquid 1200 chromatography (RP -HPLC) using an increasing gradient of 1% buffer B (90% 1201 acetonitrile,0.05% TFA) in buffer A (0.05% TFA) over an 80 min period (Phenomenex Jupiter 1202 300 Å, 10 µm, 250 x 21.2 mM). Analysis was performed using electrospray mass spectrometry 1203 (ESI-MS) (AB SCIEX API 2000) to identify fractions containing mass/charge ratios that 1204 matched the desired product. Purity was determined using analytical RP-HPLC (Agilent, 300 1205 Å, 5 µm, 150 x 2.1 mM) with all peptides purified until >95% purity. C5a was synthesised as 1206 previously described91. 1207 mAb4C8 production and Fab4C8 generation 1208 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 46 C5aR2-specific monoclonal antibodies producing hybridoma clones (mAb4C8) were obtained 1209 as a generous gift from Monash University. The frozen clones were revived in Dulbecco’s 1210 Modified Eagle Medium (DMEM) supplemented with 10% FBS and cultured at 37 °C in a 5% 1211 humidified CO2 incubator. Once the cells reached optimal density, approximately, 40 million 1212 cells were seeded in CELLine bioreactor (DWK Life Sciences; Cat. No: WCL -1000) using 1213 Hybridoma SFM (serum free medium) (Gibco, Cat. No: 12045 -076) to facilitate high -yield 1214 antibody production. The culture was maintained at 37 °C in a 5% CO ₂ humidified incubator, 1215 with supernatant was harvested weekly for antibody collection. Fresh Hybridoma SFM was 1216 replenished at each harvest to sustain continuous cell proliferation and monoclonal antibody 1217 secretion. The supernatant from each harvest is flash frozen in liquid nitrogen and stored at 1218 -80 °C till further use. 1219 For antibody purification, the harvested supernatant was thawed and subjected to 1220 centrifugation at 22,000 x g for 20 min to remove residual cellular debris and sequentially 1221 filtered through 0.45 µ pre-filters followed by 0.22 µ filters to ensure the removal of particulates. 1222 To facilitate mAb binding to protein A affinity resin (MabSelectTM, Cytiva Cat. No: 17519902), 1223 the supernatant was buffered with 2 M Na 2HPO4, pH 8.0, prior to loading on pre-equilibrated 1224 gravity flow columns containing ProteinA beads. The loading was carried out at a constant 1225 flow rate of 0.5 mL/min to maximize antibody recovery, following this the column was washed 1226 with 1x PBS, pH 7.4 (self -prepared) to remove unbound contaminants. The presence of 1227 antibody in wash fractions was monitored by measuring absorbance at 280 nm (A280) for IgG 1228 using NanoDrop (Thermo Fisher Scientific) and carried out till the absorbance reached a 1229 minimal baseline value. Subsequently, the column was washed with 100 mM NaCl to displace 1230 loosely bound non -specific proteins followed by washing with 1x PBS. The bound mAb4C8 1231 was eluted under low pH buffer condition using 100 mM NaH 2PO4, pH 2.5, and eluate was 1232 immediately neutralised with 1M Na2HPO4, pH 8.0. The elution was carried out till A280 for IgG 1233 minimises to ≤0.01, indicating the complete antibody recovery. The purified mAb4C8 was 1234 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 47 dialysed against buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl for 16 h and flash 1235 frozen in liquid nitrogen and stored at -80 °C until further use. 1236 To generate antigen -binding fragments (Fab), purified mAb4C8 was digested using 1237 papain. 10 mg mAb4C8 was concentrated to 20 - to 25-fold using a Cytiva Vivaspin 100 kDa 1238 MWCO centrifugal device (Cat. No: 28932363). Papain digestion was performed using a crude 1239 papain extract derived from Carica papaya (Sigma, Cat. No: P375 -25G). The extract was 1240 solubilised in papain digestion buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM EDTA, 1241 50 mM L-cysteine) and vortexed until completely dissolved. Insoluble debris was removed by 1242 centrifugation at 14,000 rpm for 10 min and the resulting supernatant was used as the enzyme 1243 source. The digestion was set up by incubating 10 mg of mAb4C8 with papain at final enzyme 1244 concentration of 50 µg/mg of mAb at 37 °C for 3 h. The reaction was irreversibly quenched 1245 with 50 mM iodoacetamide. 1246 To separate the Fab and Fc fragments, the reaction mixture was loaded onto a pre -1247 equilibrated HiLoadTM 16/600 Superdex™ 200 pg (Cytiva, Cat. No: 28989335) for SEC. The 1248 purified Fab fragment was eluted in a buffer containing 20 mM HEPES, pH 7.4 and 150 mM 1249 NaCl, pooled, flash-frozen in liquid nitrogen and stored in 10% glycerol at -80 °C for further 1250 experiments. 1251 Complexing of Fab4C8-bound Apo/C5apep/EP54/R8Y-C5aR2 1252 Purified C5aR2 was mixed with 2-fold molar excess SEC purified Fab4C8 with or without either 1253 C5apep or EP54 or R8Y at a final concentration of 10 µM to form Fab4C8 -bound Apo and 1254 peptide-bound C5aR2 complexes, respectively. The reaction mix was allowed for complexing 1255 in constant tumbling conditions at room temperature. The complex was separated by injecting 1256 the reaction mix in a Superose™ 6 Increase 10/300GL column (Cytiva, Cat. No: 29091596), 1257 equilibrated with SEC buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 0.01% L -MNG, 0.001% 1258 CHS, 1 µM either EP54/C5a pep/R8Y) and the peak fractions were pooled together, 1259 supplemented with either EP54/C5apep/R8Y at a final concentration of 10 µM and concentrated 1260 to 5-20 mg/mL concentration in a 100 kDa centrifugal device. 1261 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 48 Ib30 reactivity 1262 Agonist induced Ib30 reactivity downstream to hC5aR2 and mC5aR2 was measured by 1263 following the same protocol as described for NanoBiT -based ꞵarr1/2 recruitment and as 1264 discussed previously 104,105. HEK -293T cells were transiently transfected with 5 µg of Ib30 1265 tagged with N-terminal LgBiT fragment, 2 µg of ꞵarr1 tagged with C-terminal SmBiT fragment 1266 and untagged 3 µg of either hC5aR2 or mC5aR2 cloned in pcDNA3.1. 1267 Quantification and statistical analysis 1268 GraphPad Prism 10.3.1 software was used to plot and analyze all the functional data 1269 presented in this manuscript, and all the relevant details such as number of replicates, data 1270 normalization, mean ± SEM, and statistical analyses are mentioned in the corresponding 1271 figure legends. 1272

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It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint 0 60 0 60 0 60 0 60 0 5 10 15 Vehicle + C5a Vehicle + C5a-d-Arg PMX205 + C5a PMX205 + C5a-d-Arg ✱✱✱✱ ns No. of neutrophils (109/L) -12 -10 -8 -6 0 5 10 15 hC5a hC5a-d-Arg -12 -10 -8 -6 0 2 4 6 8 mC5a mC5a-d-Arg 0 40 80 120 -11 -9 -7 mC5a mC5a-d-Arg 0 40 80 120 -11 -9 -7 phC5a phC5a-d-Arg 0 40 80 120 -12 -10 -8 -6 phC5a phC5a-d-Arg Ca2+ flux assay βarr2 recruitment Phospho-ERK response Fold normalized B 10 100 10 100 0 2 4 6 8 ✱✱ ✱✱ B 1 10 100 1 10 100 0 10 20 30 ✱✱ ✱ ✱✱ Blank phC5a-d-ArgphC5a C5a-d-Arg C5a C5aR1 Neutrophil migration in mouseR Time (min) S Recombinant ligands IL-8 release B 1 10 100 1 10 100 0 10 20 30 ns ✱ ✱ B 1 10 100 1 10 100 0 2 4 6 8 ✱ ✱ Blank hC5a hC5a-d-Arg PMN migration 0 40 80 120 -12 -10 -8 -6 hC5a hC5a-d-Arg Ca2+ flux assay Phospho-ERK response 0 40 80 120 -12 -10 -8 -6 mC5a-d-Arg mC5a -14 -12 -10 -8 -6 0 30 60 90 120 hC5a hC5a-d-Arg -14 -12 -10 -8 -6 0 30 60 90 120 hC5a hC5a-d-Arg -14 -12 -10 -8 -6 0 30 60 90 120 mC5a mC5a-d-Arg -14 -12 -10 -8 -6 0 30 60 90 120 mC5a-d-ArgmC5a Gαo dissociation cAMP inhibition mC5aR1 -12 -10 -8 -6 0 2 4 6 8 mC5a mC5a-d-Arg -12 -10 -8 -6 0 2 4 6 8 hC5a hC5a-d-Arg -12 -10 -8 -6 0 10 20 30 40 hC5a hC5a-d-Arg 0.0 0.5 1.0 1.5 -12 -10 -8 -6 0 10 20 30 mC5a mC5a-d-Arg 0 1 2hC5aR1 βarr1 recruitment βarr2 recruitment mC5aR1hC5aR1 log [agonist] (M) % normalized log [agonist] (M) Fold- normalized log [agonist] (M) % normalized Fold normalized % normalized [Agonist] (nM) Fold normalized Plasma-derived ligands log [agonist] (M) [Agonist] (nM) BRET ratio % normalized 0 40 80 120 -12 -10 -8 -6 phC5a-d-Arg phC5a Fold normalized 0 40 80 120 -11 -9 -7 hC5a hC5a-d-Arg A B C D E F G H I J K L M N O 0.0 0.1 0.2 -10 -8 -6 phC5a phC5a-d-Arg 0.25 P Q HMDM BMDM HEK -293 cells mC5a - mC5aR1 mC5a - d - Arg - mC5aR1 Structural snapshots of mC5aR1 bound to mC5a and mC5 -d-Arg mC5a-mC5aR1 mC5a-d-Arg-mC5aR1 Tyr6.51 Arg4.64 Arg5.42 Asn100ECL1 Asn7.35 G73 L72 Q71 Tyr6.51 Arg4.64 Arg5.42 Asn100ECL1 Asn7.35 G73L72 Q71 R74 G-protein β-arrestin .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint BioRender Figure 1: Naturally encoded ligand bias at complement anaphylatoxin receptor for C5a (C5aR1) (A) Schematic illustration representing C5a-d-Arg mediated ligand bias at C5aR1. Schematic was prepared in BioRender. (B) Heterotrimeric GoA dissociation (upper panel) and cAMP inhibition (lower panel) was measured as a functional readout of human (h) and mouse (m) C5aR1 activation by (h/m) C5a and (h/m) C5a-d-Arg, employing NanoBiT-based enzyme complementation assay and GloSensor assay, respectively. Data (mean ± SEM) represent n=3-4 independent experiments. (C) βarr1/2 recruitment downstream to (h/m) C5aR1 in response to (h/m) C5a and (h/m) C5a-d-Arg measured by NanoBiT assay. Data (mean ± SEM) represent n=3 independent experiments, fold normalized with respect to response recorded for lowest ligand dose. The inset within the upper panel displays the bias plot representing the G-protein bias encoded by C5a-d-Arg. C5aR1 mediated signaling in response to recombinant (D-K) and human plasma-derived (L-Q) C5a and C5a-d-Arg showing C5a-d-Arg preferentially mediates G-protein signaling upon activation. (D-E) Ligand induced intracellular calcium mobilization in human monocyte derived macrophages (HMDMs) (upper panel, in response to hC5a and hC5a-d-Arg) and mouse bone marrow derived macrophage (BMDM) (lower panel, in response to mC5a and mC5a-d-Arg) was monitored using the calcium dye Fluo-4 NW for 100 seconds, with ligand added at 16 seconds. Changes in fluorescence were normalized to the maximum ligand-induced response. Data represent mean ± SEM (n=3), independent donors. (F-I) ERK phosphorylation using SRE-based reporter assay in HEK-293 cells (F-G) and AlphaLISA Surefire-Ultra p-ERK1/2 kit in HMDM (H) /BMDM (I). Data was fold-normalized to the lowest ligand concentration for SRF-RE reporter assay. Data represent mean ± SEM (n≥4), independent experiments. (J) IL-8 release in HMDMs in response to hC5a and hC5a-d-Arg. Cells were incubated with respective ligands for 24 h prior to supernatant collection. Data was analysed using repeated measures one-way ANOVA followed by Dunnette’s post-hoc test, comparing each condition to the unstimulated control (Blank). Significant IL-8 release were observed at 100 nM for both hC5a (adjusted p=0.0106) and hC5a-d-Arg (adjusted p= 0.0352). Data represent mean ± SEM (n≥4), independent experiments. (K) Ligand-induced human polymorphonuclear neutrophils (PMN) chemotaxis was assessed using the Corning® FluoroBlok system, with migration quantified at 20 minutes post-ligand addition and normalized to the medium-only treated cells (fold-baseline, n = 3 independent donors) (ANOVA, p < 0.05*, p < 0.01**, p < 0.001***). (L) Ligand induced Calcium mobilization in HMDM in response to plasma-derived hC5a and hC5a-d-Arg. (M) βarr2 recruitment in response to increasing ligand concentration downstream to C5aR1 measured by BRET-based assay in HEK-293 cells. The ligand- induced BRET ratio following ligand stimulation at 40 minutes, normalized with respect to that of lowest ligand dose. (N-O) ERK- phosphorylation assay (similar to panel H-I) in HMDM (N) and BMDM (O) in response to varying ligand doses. Data represent mean ± SEM (n ≥ 4), independent experiments. (P-Q) IL-8 release (P) and PMN migration (Q) in response to plasma-derived hC5a and hC5a-d-Arg (undertaken similar to panel J-K, respectively). Data represent fold-baseline, n = 3 independent donors (ANOVA, p < 0.05*, p < 0.01**, p < 0.001***) (R) Neutrophil migration in mouse in response to C5a and C5a-d-Arg in the absence and presence of C5aR1-inhibitor, PMX205, measured post 60 minutes ligand injection to mouse. Data represent n=4 independent experiments, and analysis was carried out by using two-way ANOVA, Tukey’s multiple comparisons (p<0.0001**** and ns = not significant) (S) Structural snapshots of mC5a and mC5a-d-Arg bound mC5aR1-Gαoβγ-ScFv16 complexes determined by cryo-EM at a global resolution of 3.15 Å and 3.13 Å, respectively (Gαo, yellow, Gβ, magenta Gγ, turquoise, ScFv16, gray). The right panel showing the interaction of carboxyl-terminal residues of mC5a and mC5a-d-Arg with the residues present in orthosteric binding cavity of mC5aR1. .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint C5a-d-Arg-C5aR1 C5a-C5aR1 p324/327 0 50 100 150 ✱✱✱ ns ✱✱✱ 0 50 100 150 ns ns ns 0 50 100 150 ✱✱✱✱ ✱✱✱✱ ✱✱ 0 50 100 150 ✱✱✱✱ ✱✱✱✱ ns p332/334GRK2 GRK6 % normalized GRK recruitment (receptor-SmBiT) hC5aR1 -10 -9 -8 -7 -6 0.8 1.0 1.2 1.4 V hC5a hC5a-d-Arg -10 -9 -8 -7 -6 0.8 1.2 1.6 2.0 V hC5a hC5a-d-Arg -10 -9 -8 -7 -6 0.8 1.0 1.2 1.4 V mC5a mC5a-d-Arg -10 -9 -8 -7 -6 0.8 1.0 1.2 1.4 V mC5a mC5a-d-Arg -10 -9 -8 -7 -6 0.8 1.2 1.6 2.0 V mC5a mC5a-d-Arg -10 -9 -8 -7 -6 0 1 2 3 V mC5a mC5a-d-Arg -10 -9 -8 -7 -6 0.8 1.0 1.2 1.4 V hC5a hC5a-d-Arg -10 -9 -8 -7 -6 0.8 1.0 1.2 1.4 V hC5a hC5a-d-Arg GRK2GRK3GRK5GRK6 Fold normalized log [agonist] (M) Deuterium uptake (%) D2O incubation time (s) mC5a - mC5aR1 mC5a - d - Arg - mC5aR1 TM7 H8 TM1 TM2 A hC5a-hC5aR1 hC5a-d-Arg-hC5aR1 Arg 4.64 Arg 5.42 Asp 7.35 Arg 4.64 Arg 5.42 Asp 7.35 Arg4.64 32% Asp7.35 Arg5.42 90% Arg5.42 57% Arg4.64 70% Asp7.35 95% Key ResiduesSimulation data -12 -10 -8 -6 0 5 10 15 20 25 WT R4.64A R5.42A D7.35A -12 -10 -8 -6 0 5 10 15 20 25 WT R4.64A R5.42A D7.35A % normalized -14 -12 -10 -8 -6 30 60 90 120 WT R4.64A R5.42A D7.35A -14 -12 -10 -8 -6 30 60 90 120 WT R4.64A R5.42A D7.35A cAMP responseβarr1 recruitment Fold normalized log [hC5a-d-Arg](M)log [hC5a](M) B C hC5aR1 hC5a - hC5aR1 hC5a - d - Arg - hC5aR1 Ile2.64 Phe7.28 TM7 dynamics Q71 R74 Ser7.36 Asn7.35 Q71 Asn7.35 Ser7.36 TM7 mC5a and mC5a-d-Arg TM7 contactsD HDX-MS analysis H I mC5aR1 Receptor phosphorylation hC5a hC5a-d-Arg M __ _ ++_ __ +_ _ + 54 43 54 43 IB:p332/334IB:p324/327 IB:FLAG M + _ +_ _ _ GRK2 43 M _ +_ _ _ + 54 43 54 GRK6 mC5a mC5a-d-Arg IB:FLAG M hC5aR1 mC5aR1 hC5aR1mC5aR1 Helix 8 Helix 8 movement E hC5a - hC5aR1 hC5a - d - Arg - hC5aR1 NTSR1 - GRK2 mC5a - mC5aR1 mC5a - d - Arg - mC5aR1 NTSR1 - GRK2 Helix 8 Apo-mC5aR1 mC5a-mC5aR1 mC5a-d-Arg-mC5aR1 hC5a hC5a-d-Arg D2O incubation time (s) Deuterium uptake (%) mC5a-mC5aR1 mC5a-d-Arg-mC5aR1 F G 10 100 1000 10000 20 30 40 50 60 277-286: TM7 *** 10 100 1000 10000 20 30 40 50 60 33-39: N-term of TM1 ** 10 100 1000 10000 20 30 40 50 60 72-86: TM2 *** *** 10 100 1000 10000 20 30 40 50 60 313-324: H8 ** .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint Figure 2: Mechanistic insights into G-protein bias driven by C5a-d-Arg (A) Structural snapshots illustrating key interactions of hC5a-hC5aR1 (PDB: 8IA2) and hC5a-d-Arg-hC5aR1 (PDB: 8JZZ). Models are represented in tube helices with hC5a in royal blue, hC5a-d-Arg in lime green, hC5a-bound hC5aR1 in brick-red and hC5a-d-Arg-bound hC5aR1 in dodger-blue. Molecular dynamic simulation (MDS) (lower panel) highlighting the percentage involvement of orthosteric binding residues with hC5a and hC5a-d-Arg. (B-C) cAMP inhibition (B) and βarr1 recruitment (C) measured downstream of hC5aR1 mutants (R4.64A, R5.42A, D7.35A), rationally designed based on MDS studies. Data represents mean ± SEM (n=3), independent experiments. (D) Cartoon representation comparing helix 8 movement in hC5a-hC5aR1 (8IA2) Vs. hC5a-d-Arg-hC5aR1 (8JZZ) and mC5a- mC5aR1 Vs. mC5a-d-Arg-mC5aR1 structures with respect to NTSR1-GRK2 (PDB: 8JPF) structure, revealing distinct positioning of helix 8 in C5a-d-Arg-bound C5aR1, potentially occluding GRK docking site in the receptor. (E) Structure-based simulation studies comparing C5a and C5a-d-Arg activation of C5aR1 highlighting a key difference in TM7 conformation, measured by the distance between Ile2.64 and Phe7.28 (indicated by black dots) in C5a-d-Arg and C5a-activated mutants. (F) Structural superposition of mC5a and mC5a-d-Arg-bound mC5aR1 highlighting the loss of hydrogen bonds with Ser7.36 and Asn7.32 of TM7 when bound to C5a-d-Arg. (G) Structural superposition of mC5a-mC5aR1 and mC5a-d-Arg-mC5aR1 (left-panel) highlighting the transmembrane regions (TM) undergoing reduced HDX. HDX-MS analysis (right) of mC5aR1 highlighted regions (left) undergoing significant decrease in deuterium uptake upon incubation either with ligand (mC5a/mC5a-d-Arg) or without ligand (Apo-mC5aR1). Statistical analysis was performed by applying one-way ANOVA followed by Tukey’s multiple comparisons test. (p<0.001***, p<0.01**). (H) NanoBiT-assay showing GRK recruitment downstream to (h/m) C5aR1 in response to (h/m) C5a and C5a-d-Arg.Data represents mean ± SEM (n=3), independent experiments, normalized with lowest ligand concentration considered as 1. (I) Phosphorylation detection via pIMAGO kit (upper panel) and phosphorylation site-specific antibodies (lower panel) to assess the receptor phosphorylation following ligand stimulation. Representative blots and densitometric analysis are shown below (green- mC5a, pink – mC5a-d-Arg, blue – hC5a and maroon – hC5a-d-Arg. Densitometric plots represent mean ± SEM (n = 3-4), independent experiments, with stimulated condition normalized with respect to unstimulated condition which is considered as 100% (p<0.0001****, p=0.0001***, p=0.0013**). .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint C5a C-terminal interaction with C5aR2 and C5aR1 A C - terminal of C5a Orthosteric binding pocket of C5aR2 E 5.35 E 7.35 R 4.64 V188 C - terminal of C5a Orthosteric binding pocket of C5aR1 D 7.35 R 4.64 S 2.63 H100 Y 6.51 M 6.58 V190 D 191 T 6.59 H67 K68 D69 M70 Q71 G73 R74 H67 K68 D69 M70 Q71 G73 R74 hC5a - h C5aR2 PMX53 - hC5aR1 TM7 Helix 8 4Å 6.5Å TM6 ECL2 Helix4 Helix3 Helix2 N - terminus C5a-d-Arg C5a C5aR2 biased signalling -12 -10 -8 -6 0 2 4 6 hC5a hC5a-d-Arg -12 -10 -8 -6 0 2 4 6 mC5a mC5a-d-Arg -12 -10 -8 -6 0 1 2 3 hC5a hC5a-d-Arg -12 -10 -8 -6 0 1 2 3 mC5a mC5a-d-Arg mC5aR2 Fold normalized log [agonist] (M) βarr1 recruitment hC5aR2 Overall architecture of hC5a-hC5aR2 B C J βarr2 recruitment C5aR2 hC5a TM movements and helix 8 orientation 10 100 1000 10000 0 20 40 60 * * * * 10 100 1000 10000 0 20 40 60 * * * * 10 100 1000 10000 0 20 40 60 * * * * 10 100 1000 10000 0 5 10 15 20 * * 10 100 1000 10000 0 20 40 60 * * * * 10 100 1000 10000 0 20 40 60 * * * * 10 100 1000 10000 0 15 30 45 * * * 30AIDPLRVAPLPLYAAIF45 134FLALGPAWWSTVQ146 165LTVPSAI171 245CWAPYHLLGLV255 62RANISHKDMQLGR74 257TVAAPNSAL265 266LARALRAEPLIVG278 Deuterium uptake (%) D2O incubation time(s) D2O incubation time(s) 9IAAKYKHSVVAK20 10 100 1000 10000 0 15 30 45 * * * * (i) (ii) (iii) (iv) (v) (vi) (vii) (viii) Apo-C5aR2 C5a-C5aR2 Apo-C5aR2 C5a-C5aR2 (iv) (ii) (iii) ( i ) (v) (vi) (vii) (viii) C5a-C5aR2 ( i ) N - term (ii) ICL2 (iii) TM4 (iv) TM6 (v) ECL3 (vi) TM7 (vii) Helix 2/3 of C5a (viii) C - terminus of C5a I2.59 L2.60 S/P2.63 I2.64 H100/G90 W88/100 P3.29 I3.32 L3.33 M3.36 S4.60 R4.64 V176/R174 R178/H176 Y181/H179 F182/180 L187/Q185 C188/186 G189/V187 V190/188 D191/189 Y192/190 H194/G191 E5.35 R5.42 Y6.51 T/L6.54 G6.55 M/L6.58 S/T6.59 L/A6.61 S271/N262 S7.25 P7.26 K7.29 K/L7.32 D/E7.35 hC5aR1 hC5aR2 0 1 2 TM2 ECL1 TM3 TM4 ECL2 TM5 TM6 ECL3 TM7 D Two-site binding ECL2 shift hC5a - hC5aR1 hC5a - h C5aR2 E F G Two site binding Core - domain Site 1 Site 2 C - term C5a-interactions HDX-MS Analysis I hC5aR2 ECL2 D25 L24 V21 L22 T24 D27 T29 C5aR2 C5aR1 N-term interaction H N-terminus shift RMSD ~ 1Å Deuterium uptake (%) G-protein β-arrestin .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint Figure 3: Functional and structural insights into C5a binding to C5aR2 (A) C5aR2 selectively signals through β-arrestin upon ligand binding, remains inactive in terms of G-protein signaling. Schematic was prepared in BioRender. (B) βarr1/2 recruitment downstream to (h/m) C5aR2 in response to (h/m) C5a and C5a-d-Arg measured by NanoBiT-assay in HEK- 293 cells. The data represents mean ± SEM (n=6), independent experiments where fold normalization is carried out by normalizing the luminescence values for each ligand dose with respect to the smallest ligand dose taken as 1. (C) Cryo-EM density map of Trx-C5a-bound C5aR2 at 3.8 Å and structural snapshot representing overall seven-transmembrane architecture of C5a-bound C5aR2 (show in tube helices) with ECL2 shown in ribbon representation. (D) Superposition of PMX53-bound C5aR1 (PDB:6C1R) and C5a-bound C5aR2, comparing transmembrane (TM6 and TM7) and helix 8 orientation in C5aR2 upon activation. (E) Superposition of C5a-bound C5aR1 (PDB:8IA2) and C5a-bound C5aR2, comparing differential interactions of C5a with C5aR1 and C5aR2. Upper-right panel showing an upward shift in the N-terminus of C5aR2 to establish contact with helix 2 of C5a. Lower- right panel depicting shift in ECL2 of C5aR2 towards cytoplasmic cavity with respect to C5aR1 ECL2. (F) Two-site binding of C5a (shown in surface) and C5aR2 (shown in tube helices). (G-H) Schematic representation of C5a core helices interaction with N-terminus (G) and carboxyl-terminal terminal interaction with C5aR2 and C5aR1 orthosteric pocket residues (H). Black and red dotted lines represent hydrogen bonds and salt-bridge interactions, respectively. (I) Heat map displaying global interaction map of C5a-bound to C5aR1 and C5aR2. Each column represents an analogous residue position in C5aR1/2, where an interaction with C5a may be mapped. TM-helix residues have been numbered according to the Ballesteros-Weinstein (BW) numbering scheme and those in the N-term or loops have been numbered according to their order of occurrence in the receptor. To determine conserved residue positions, transmembrane helix residues were looked up from the GPCRdb (GPCRdb.org). If the residues at a particular BW-position are different across the receptors, they have been indicated. For example, at the BW 2.63 position, because C5aR1 has S and C5aR2 has P, it has been indicated as S/P2.63. For loops, where the GPCRdb did not report corresponding residues, they were inferred from the structural superposition of hC5a-bound hC5aR1 (PDB 8IA2) and hC5a-bound C5aR2. Similar to TM-residue numbering, any difference in the residue or its number at a corresponding position have been indicated. Heat-map scale shows whether a particular residue of C5aR1 or C5aR2 is involved in the interaction with C5a. If a particular residue from both the receptors is involved in interaction with C5a, it is given a score of 2, if it exists in only one receptor, it has given a score of 1 and the corresponding interaction for the other receptor is given a score of 0. Scores are encoded in colors and visualized as heat-maps. (J) Cartoon representation of C5a-bound C5aR2 highlighting the regions undergoing significant reduction in deuterium uptake. (i-viii) Deuterium uptake plots of selected peptides of C5aR2 and C5a (brown: Apo-C5aR2, turquoise: C5a-C5aR2). Results were derived from three independent experiments. The statistical significance of the differences was determined using Student’s t-test (*p< 0.05). Data are presented as mean ± standard error of the mean. * Indicates statistically significant difference between alone and complex, respectively. .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint Biochemical characterisation Protomer 1 Protomer 2 Cys 306 Cys 316 Cys 316 Cys 306 Disulfide bond Helix 8 Helix 8 90 ° Hydrophobic packing Protomer 1 Protomer 2 Phe 1.43 Phe 1.43 Leu 1.44 Leu 1.44 TM1 TM1 Helix 8 TM1 Dimeric interface of mC5aR2 D C 0 0 100 200 300 400 500 8 9 10 11 12 13 14 15 Elution volume (mL) Absorbance at 280 nm (mAU) mC5aR2 hC5aR2 Dimer Monomer A mC5a-d-Arg-mC5aR2 (Dimer) mC5aR2 mC5a - d - Arg mC5aR2 B Two-site binding K71 K68 L75 G73 G76 R74 L72 Q74 Q71 V73 M70 P72 D69 P69 S66 H70 H67 Core - domain shift hC5a mC5a - d - Arg Orthosteric pocket H1 H2 H4 H3 Overall-ligand positioning C - terminal of hC5a Orthosteric binding pocket of hC5aR2 E 5.35 E 7.35 R 4.64 V188 T 6.59 H67 K68 D69 M70 Q71 G73 R74 Orthosteric binding pocket of mC5aR2 Binding modes of C5a and C5a-d-Arg G C - terminal of mC5a - d - Arg R 5.42 R68 V73 H70 G76 R 4.64 L75 R 6.55 Q74 E 7.35 K71 E 5.35 I 6.58 G193 I194 RMSD map RMSD 210 hC5a - hC5aR2 mC5a - d - Arg - mC5aR2 Helix 8 TM1 ICL3 ICL2 Site 2 mC5aR2 Site 1 Site 2 hC5aR2 hC5a mC5a - d - Arg E F Protomer 1 Protomer 2 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint Figure 4. Molecular insights into C5a and C5a-d-Arg binding to C5aR2. (A) Size-exclusion chromatography profile of the purified human and mouse C5aR2, revealing a substantial dimeric population for the mouse receptor. (B) Structural snapshots of cryo-EM densities and atomic models of mC5a-d-Arg-mC5aR2 complex. (C) Structural comparison of the human C5aR2 and mouse C5aR2 using superimposition and RMSD across the entire receptor mapped onto the mouse C5aR2. (D) Dimeric interface of the mC5aR2 indicating TM1-TM1 interface with the involvement of key residues, and H8-H8 interface mediated by two disulfide bridges between the indicated residues. (E) Structural snapshots revealing two-site binding mechanism of C5a-C5aR2 and mC5a-d-Arg-mC5aR2 (F) Superposition of hC5a and mC5a-d-Arg obtained from the hC5a-hC5aR2 and mC5a-d-Arg-mC5aR2 structures, showing overall ligand positioning, core-domain shift, C-terminus positioning in C5aR2 orthosteric pocket and terminal G76 of mC5a-d-Arg occupying similar positioning as that of R74 in hC5a. (G) Schematic representation of C-terminal residues of hC5a and mC5a-d-Arg interacting with different residues lining orthosteric pocket of hC5aR2 and mC5aR2 highlighting extensive interaction network of C5a and C5a-d-Arg (black dashed lines indicate hydrogen bonds, red-dashed lines indicate salt-bridges. .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint EP54 EP67 C5apep EP141 P32 P59 R8Y -YSFKPMPL[DAL]R -YSFKDMP[NME][DAL]R -[MEA]KP[ZAL][ZAL][DAR] -WWGKKYRASKLGLAR -Ac-RHYPYWR-OH -Ac-LIRLWR-OH -Ac-YKPLGL[DAL]Y-OH C5a-derived C3a-derived NME: N-methyl leucine, MEA: N-methyl phenylalanine, DAL: D-alanine, DAR: D- arginine, ZAL: β-cyclohexyl-L-alanine C5aR2- selective Peptide agonists Fold normalized -14 -12 -10 -8 -6 -4 0 50 100 150 [ Tyr8]BM2020-8 [ Tyr1,8]BM2020-8(R8Y) C5a Discovery of R8Y % normalized log [agonist] (M) U -6 -5 1 2 3 4 U -6 -5 hC5a EP54 EP67 EP141 C5apep P32 P59 hC5aR2 mC5aR2 log [agonist] (M) U -7 -6 -5 1.0 1.2 1.4 1.6 1.8 2.0 U -7 -6 -5 C3aR C5aR1 C5aR2 βarr1 recruitment βarr2 recruitment log [R8Y] (M) U -6-7 U -6-7 U -6-7 0 50 100 150 ns ns ns C3aR βarr1 recruitment U -6-7 U -6-7 U -6-7 0 50 100 150 ns ns ns C5aR1 U -6-7 U -6-7 U -6-7 0 50 100 150 ns ns ns U -6-7 U -6-7 U -6-7 0 50 100 150 ns ✱ ✱ U -5-6 U -5-6 U -5-6 0 50 100 150 ns ✱✱✱✱ ns U -6-7 U -6-7 U -6-7 0 50 100 150 ns ✱✱✱✱ ✱✱✱✱ U -5-6 U -5-6 U -5-6 0 50 100 150 ns ✱✱✱✱ ns U -5-6 U -5-6 U -5-6 0 50 100 150 ns ✱✱✱✱ ✱✱✱✱ log [agonist] (M) C5aR2 -12 -10 -8 -6 -4 0 50 100 150 R8Y 10μM 4C8 + R8Y log [R8Y] (M) -12 -10 -8 -6 0 50 100 150 hC5a 1μM 4C8 + hC5a log [hC5a] (M) βarr2 recruitment -12 -10 -8 -6 -4 0.5 1.0 1.5 2.0 2.5 hC3aR hC5aR1 hC5aR2 R8Y βarr1 recruitment Fold normalized -12 -10 -8 -6 -4 0 1 2 3 4 hC5a R8Y -12 -10 -8 -6 -4 0 1 2 3 4 mC5a R8Y hC5aR2 mC5aR2 C5aR2 % normalized log [hC3a] (M) log [hC5a] (M) log [hC5a-d-Arg] (M) log [hC5a] (M) log [hC5a-d-Arg] (M) log [C5apep] (M) log [EP54] (M) log [R8Y] (M) Ligand only mAb4C8 only mAb4C8 pre -incubation (30 minutes) + Ligand stimulation A B C D E F Fab4C8-R8Y-C5aR2Fab4C8-EP54-C5aR2 Fab4C8-C5apep-C5aR2G C5aR2 EP54 C5aR2 R8Y C5aR2 C5a pep Fab4C8-Apo-C5aR2 .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint Figure 5: Comprehensive characterization of C5aR2-specific peptide ligands and previously discovered C5aR2-blocking antibody mAb4C8. (A) Sequence of small peptide ligands known to activate C3aR and C5aR1 along with C5aR2-selective peptides, P32 and P59 and novel decapeptide, R8Y. (B) Screening of previously published peptides to assess agonistic activity on human and mouse C5aR2 measured by βarr1 recruitment using NanoBiT-based assay. The data represents mean values for two independent experiments. The heat map shows fold normalized values for two different ligand doses. The raw counts for ligand stimulated conditions were normalized with respect to unstimulated conditions taken as 1. (C) βarr2 recruitment downstream to C5aR2 in response to the novel peptide, R8Y, rationally deigned by modifying BM2020-8 peptide backbone. For measuring βarr2 recruitment, BRET-based luminescence assay was employed and % normalization was carried out by normalizing response for varying ligand doses with respect to that of the highest ligand dose taken as 100. (D) βarr1/2 recruitment to investigate selectivity profile of R8Y in activating C3aR, C5aR1 and C5aR2 at two ligand doses. NanoBiT- based assay was employed in HEK-293 cells transiently transfected with either C3aR or C5aR1 or C5aR2. Heat map showing mean values for three independent experiments, normalized with respect to unstimulated condition considered as 1. (E) Dose-response curve showing R8Y-induced βarr1 recruitment downstream to hC3aR/hC5aR1/hC5aR2/mC5aR2 highlighting its species-specific and C5aR2-selective nature. (F) Characterization of mAb4C8 by measuring its agonistic and antagonistic properties on C3aR, C5aR1 and C5aR2. NanoBiT- based βarr1 recruitment was performed under three conditions, viz. native ligand stimulated (in blue), mAb4C8 stimulated (to assess agonistic properties; in red) and pre-incubation of mAb4C8 for 30 minutes prior to induction with ligand doses (to assess antagonistic properties; in olive-green). The assay displays antagonistic activity of mAb4C8, selectively for C5aR2, marked by severe reduction in C5a and C5a-d-Arg-mediated βarr1 recruitment in the presence of mAb4C8. The right-most panel displays hC5a- or R8Y-induced dose-response curve for βarr1 recruitment downstream to hC5aR2, in the presence or absence of mAb4C8, at indicated molar concentrations. Data represents mean ± SEM for three-four independent experiments. Statistical significance of the data was carried out by applying two-way ANOVA followed by Tukey’s multiple comparisons test (p<0.0001****). (G) Structural snapshots of cryo-EM density map and models of C5aR2 bound to EP54, C5apep and R8Y along with Fab4C8 or without any ligand, i.e., Apo-C5aR2. To left, EM-density map and to right, respective model map presented in cartoon style. .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint R8Y Y8 C5aR2 Arg5.42 Tyr6.51 EP54 R10 C5aR2Tyr6.51 Glu7.35 DAR6 R74 R10 Y8 R8Y EP54 C5a C5apep R74 C5a C5aR2 Tyr6.51 Glu7.35 R8Y-C5aR2 C5a- C5aR2 C5apep- C5aR2 EP54-C5aR2 W6.48 8.3Å W6.48 9.3Å W6.48 7.7Å R8Y-C5aR2EP54-C5aR2 C5apep-C5aR2 W6.48 9.3Å C5a-C5aR2 Terminal residues Terminal residue interaction with C5aR2 C5apep C5aR2 Interaction conservation map of ligands activating C5aR2 Across receptor residue conservation of EP54 and C5a pep Overall positioning of peptide ligands in orthosteric binding pocket of C5aR2A B C D E C5apep-C5aR2 C5apep-hC5aR1 DAR6 K2 MEA1 DAR6 K2 MEA1 4.6Ǻ EP54-C5aR2 EP54-hC5aR1 EP54 EP54 EP54 EP54-hC3R Arg4.64 Arg5.42 Tyr3.37 Tyr6.51 DAR6 H F G I F/I/I2.59 S/L/L2.60 H/S/P2.63 L/I/I2.64 Q/H/H2.67 G86/H100/G90 W88/102/100 P3.29 I3.32 V/L/L3.33 M3.36 F/Y/Y3.37 V/S/S4.60 R4.64 E162/V176/R174 F164/R178/H176 T166/E180/E178 D167/Y181/H179 N168/F182/F180 R171/L187/Q185 C172/188/186 G173/G189/V187 Y174/V190/V188 -/D191/D189 -/Y192/Y190 -/H194/G191 V/D/S5.31 L/E/E5.35 R5.42 Y6.51 F/T/L6.54 G6.55 S/M/L6.58 L/S/T6.59 T/L/A6.61 D404/E269/A260 P405/P270/P261 E406/S271/N262 T/S/S7.25 P/P/P7.26 G/F/L7.28 K7.29 M/K/L7.32 D/D/E7.35 C/C/I7.38 I/V/V7.39 S/Y/L7.43 hC3a EP54 hC5a hC5a-d-Arg EP54 C5apep hC5a EP54 C5apep R8Y 0 2 4 6 8 10 TM4 ECL3TM6TM2 ECL1 TM3 ECL2 TM5 TM7 C3aRC5aR1C5aR2 Residue interaction map of complement receptor and agonists J F/I/I2.59 S/L/L2.60 H/S/P2.63 W88/102/100 P3.29 I3.32 V/L/L3.33 M3.36 V/S/S4.60 R4.64 E162/V176/R174 F164/R178/H176 R171/L187/Q185 C172/188/186 G173/G189/V187 Y174/V190/V188 -/D191/D189 -/H194/G191 V/D/S5.31 L/E/E5.35 R5.42 Y6.51 F/T/L6.54 G6.55 S/M/L6.58 T/L/A6.61 D404/E269/A260 P405/P270/P261 G/F/L7.28 M/K/L7.32 D/D/E7.35 C/C/I7.38 I/V/V7.39 S/Y/L7.43 C3aR C5aR1 C5aR2 0 1 2 3 EP54 TM4 ECL3TM6TM2 ECL1 TM3 ECL2 TM5 TM7 L2.60 W102/100 P3.29 I3.32 L3.33 Y3.37 S4.60 R4.64 R178/H176 L187/Q185 C188/186 G189/V187 V190/188 D191/189 Y192/190 R5.42 Y6.51 G6.55 M/L6.58 S/T6.59 L/A6.61 F/L7.28 K/L7.32 D/E7.35 V7.39 C5aR1 C5aR2 0 1 2 C5apep TM4 TM6TM2ECL1 TM3 ECL2 TM5 TM7 V21 L24 D25 I2.59 L2.60 W100 L3.28 P3.29 I3.32 L3.33 Y3.37 S4.60 R4.64 R174 H176 H179 F180 C186 V187 V188 D189 Y190 E5.35 R5.42 Y6.51 L6.54 G6.55 L6.58 T6.59 A6.61 A260 P261 N262 A7.26 A7.29 L7.32 E7.35 I7.38 V7.39 C5a EP54 C5apep R8Y 0 1 2 3 4 TM4 ECL3TM6N-term ECL1 TM3 ECL2 TM5 TM7TM2 E .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint Figure 6: Molecular insights into diverse ligand recognition by C5aR2. (A) Overall structural superposition of C5aR2 bound to C5a, EP54, C5apep, and R8Y. (B) Superposition of the C-terminal part of C5aR2-bound ligands, in the C5aR2 orthosteric pocket in the C5apep-C5aR2 structure (green background). The hook-shaped C-termini of all ligands (shown in ribbon and atom representation) fit into a similar binding cavity in the orthosteric pocket of C5aR2, shown as surface slices, with the depth of penetration of each ligand from the conserved W6.48 residue of C5aR2. (C-D) Superposition of the C-terminal residues (R74 in C5a, R10 in EP54, DAR6 in C5apep and Y8 in R8Y) of C5aR2-bound ligands showing their similar or distinct orientations in the C5aR2 orthosteric pocket (C), which leads to them engaging distinct sets of residues for interaction (D). (E) Heatmap summarizing interactions between the ligands C5a, R8Y, EP54 and C5apep with C5aR2, as inferred from the respective structural snapshots, where each row represents a ligand, and each column represents a C5aR2 residue where an interaction with a ligand may be mapped. TM-helix residues have been numbered according to the Ballesteros-Weinstein (BW) numbering scheme and those in the N-term or loops have been numbered according to their order of occurrence in the receptor. (F) Superposition of C5apep bound to C5aR1 and C5aR2 depicting slight differences in the orientation or positioning of some residues (shown by dashed straight or curved lines), although the overall conformation of the ligand backbone is maintained. (G) Heatmap comparing interactions of C5apep with C5aR1 (PDB: 9UMR ) or C5aR2. The residue numbering and analogous residue determination scheme is same as in Figure 3I. (H) Superposition of EP54 bound to either hC3aR, hC5aR1 or C5aR2, showing an overall conserved hook-like conformation, with slight shifts in the C-termini, indicated by dashed lines (I) Heatmap comparing EP54 interactions with analogous receptor residues across EP54-bound C3aR (PDB 8I95), EP54-C5aR1 (PDB: 9UMX) and EP54-C5aR2. The residue numbering and analogous residue determination scheme is same as in Figure 3I, with the inclusion of analogous positions for hC3aR as well, using the hC3a-hC3aR as a reference (PDB: 8I9L). (J) Heatmap comparing ligand-receptor interactions at analogous positions across complement receptors, viz., hC3a-hC3aR (PDB: 8I9L), EP54-hC3aR (PDB: 8I95), hC5a-hC5aR1 (PDB 8IA2), hC5a-d-Arg –hC5aR1 (PDB: 8JZZ), EP54-hC5aR1 (PDB: 9UMX), C5apep-C5aR1 (PDB: 9UMR), hC5a-C5aR2, EP54-C5aR2, C5apep-C5aR2 and R8Y-C5aR2. The residue numbering scheme is same as in Figure 3I. .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint C Cytoplasmic cavity of C5aR1 and C5aR2 Positively charged pocket Hydrophobic pocket C5aR1 C5aR2 Pocket dimension Y 7.53 C 34.53 ~2,900Å3 ICL2 TM7 F 7.53 ~4,245Å3 W 34.52 TM7 ICL2 PMX53- hC5aR1 hC5a- hC5aR1 hC5a- C5aR2 CXCL12- CXCR7 A TM6 and ICL3 shortening in C5aR2 TM 6 TM 5 TM 6 ICL3 C5a - C5aR1 C5a - C5aR2 EP54 - C5aR2 C5a pep - C5aR2 R8Y - C5aR2 C5aR1 - Gi C5aR2 hC3aR - Go CXCR2 - Go GPR109A - Gi PAF - Gi A2A - Gi 90 ° ICL3 B α 5 Helix Go Partial helix in ICL2 C5a-C5aR2 90 ° Pro138 Gly139 TM4 TM5 ICL2 TM5 α 5 Helix α N Helix Asp 3.49 Phe / Cys 3.51 Arg / Lys 3.50 TM3 TM5 C5a - C5aR1(Go) C5a - C5aR2 Disulfide Bond with TM5 TM5 TM3 C5a-C5aR2 TM5 Leu 3.50 TM3 Asp 3.49 Cys 3.51 Cys 5.57 Intra-chain disulfide bond NPxxY Vs. NPxxF Motif Tyr / Phe 7.53 α 5 Helix TM3 TM7 DRY(F) vs. DLC Motif D Arg / Lys 3.50 log [hC5a] (M) C5aR1-C5aR2C5aR2-C5aR1 Chimera design -1 4 -1 2 -1 0 -8 -6 3 0 6 0 9 0 1 2 0 C 5 a R 1 C 5 a R 2 C 5 a R 1 -R 2 C 5 a R 2 -R 1 -14 -12 -10 -8 -6 30 60 90 120 C5aR1 C5aR2 C5aR1-R2 C5aR2-R1 GαoA-dissociation cAMP response % normalized % normalized log [hC5a] (M) E .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint Figure 7: Structural insights into lack of G-protein coupling in C5aR2. (A) Surface electrostatic representation of C5aR1 and C5aR2 cytoplasmic cavity showing a positively-charged and hydrophobic cavity in C5aR1 and C5aR2, respectively. Right-most panel showing the comparison of cytoplasmic cavity of C5aR2 with respect to inactive-hC5aR1 (PMX53/Avacopan-hC5aR1; PDB: 6C1R), active- hC5aR1 (hC5a-hC5aR1-Go; PDB:8IA2), and the atypical chemokine receptor CXCR7 (CXCL12-CXCR7-FabCID24 ; PDB: 7SK5). The Gαo of hC5a-hC5aR1-Go (PDB 8IA2) structure has been docked into the cytoplasmic cavity of each receptor. The receptors are shown as surface slices and the α5 helix in ribbon representation. (B) Structural superposition of hC5a-bound hC5aR1 (PDB 8IA2) with that of C5aR2 bound to hC5a, EP54, C5apep and R8Y. Structural snapshots indicating TM5, TM6 and ICL3 shortening in C5aR2 (indicated by dashed arrow), to right. Lower-left panel indicates comparison of GPCR-Gαo/i structures, viz., hC5a-hC5aR1-Go (PDB: 8IA2), hC5a-C5aR2, CXCL8-CXCR2-Go (PDB: 8XX6), Niacin-GPR109A-Gi (PDB: 8IY9), PAF-PAFR-Gi (PDB: 8XYD) and Epinephrine-α2AAR-Gi (PDB: 9CBL) superposed on the Gαo of hC5a-C5aR1-Go (PDB 8IA2) structure showing ICL3 apposed against Gα making extensive contacts. Zoomed view (lower- right panel) of the loop and cavity shows the short ICL3 of C5aR2 is unable to dock in this cavity. (C) Conformational dynamics of ICL2 in C5aR2. Structural superposition of hC5a-bound hC5aR1 (PDB 8IA2) with that of C5aR2 bound to hC5a, EP54, C5apep and R8Y, docked on the Go of hC5a-hC5aR1-Go (PDB 8IA2) indicates that the ICL2 helix is either not formed (in EP54, C5apep or R8Y-bound C5aR2) or partially formed (in hC5a-C5aR2), in contrast to hC5aR1, where it is completely formed. This leads to loss of interactions it makes in a cavity formed by the α5 and αN helices of Gα. Inset showing the ICL2 outward swings in C5aR2 structures. Lower inset shows Pro138 and Gly139 residues in ICL2 preventing the formation of the ICL2 helix. (D) Unique NPxxY and DRY motif in C5aR2. Formation of the C5aR2 intra-helical disulfide bond (indicated in yellow-color) between Cys3.51 of the TM3 DLC-motif and Cys5.57. Lower-panel showing comparison of DRY(F) (left) and NPxxY (right) motif in hC5aR1 and C5aR2. TM7 NPxxY motif Tyr7.53 residue OH-group forms a polar contact with Arg3.50 of the DRY motif in hC5aR1, which holds Arg3.50 in position to interact with the α5-helix of Go. C5aR2, which instead has an Leu3.50 and Phe7.53, cannot form polar contacts with each other or with the α5-helix of Go. (E) Structure-guided designing of C5aR1 and C5aR2 chimeras, and dose-response curve of G-protein activation as measured by GαoA dissociation and cAMP response. Data represents mean ± SEM, n=3, independent experiments. .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint TM6TM5 A F 7 o Lack of DR(L)Y(C) motif o Disulfide bond between TM3 and TM5 restricting TM movements GP o Inability to form helix in ICL2 o Shortened TM5 and TM6 o Large and hydrophobic cytoplasmic cavity C5aR1 C5aR2 Ligand bias at C5aR1 Receptor bias at C5aR2 TM5 TM6 TM3 TM4 TM3 TM5 C5a C5aR1 C5a-d-Arg GRK Helix 8 occludes GRK docking Weak phosphorylation Robust phosphorylation C5a-d-Arg C5aR1 G-protein activation Partial βarr activation C5aR2 G-protein GRK2/3 GRK5/6 mediated phosphorylation βarr activation βγ Efficient GRK docking No G-protein coupling C5aR1 C5aR2 Figure 8: Molecular mechanism of ligand bias and receptor bias at complement anaphylatoxin receptors, C5aR1 and C5aR2. (A) Schematic summarizing the structural and functional constraints leading to C5a-d-Arg-encoded G-protein bias and intrinsic βarr bias at C5aR1 and C5aR2, respectively. Schematic was prepared in BioRender. .CC-BY-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 1, 2025. ; https://doi.org/10.1101/2025.11.01.685996doi: bioRxiv preprint