Conformational Landscape of HIV-1 Env from Closed to Fully Open

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Structural insights into these transitions have been advanced by cryo-electron tomography (cryo-ET), resolving Env structures in closed and multifarious open states within native membranes, and by cryo-electron microscopy (cryo-EM), which has provided atomic details of these states. In this study, we determined cryo-EM structures of soluble native-like Env in complex with antibody 3BC315, antibody b12, CD4, or a combination of 3BC315 and b12. These combination studies allowed us to capture previously uncharacterized HIV Env conformational states. We observed enhanced 3BC315 binding occupancy in the presence of b12 and discovered that when engaging Env, antibodies 3BC315 and b12 interact with each other directly. Moreover, we decipher the allosteric mechanisms of Env, resulting in the cooperative accommodation of 3BC315 and b12, which leads to higher occupancy and increased neutralization potency. Integrating these novel states with the literature, we establish a classification framework for symmetric and asymmetric Env states, categorizing by their degree of openness and stepwise structural rearrangements. Our findings refine the mechanistic understanding of HIV-1 Env dynamics and provide a structural roadmap for targeting dynamic Env states for more potent designs of vaccines and immunotherapeutics. Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy Biological sciences/Immunology/Infectious diseases/HIV infections Biological sciences/Microbiology/Virology/Retrovirus Biological sciences/Biochemistry/Proteins/Glycoproteins Biological sciences/Microbiology/Vaccines/Protein vaccines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The HIV-1 envelope glycoprotein trimer (Env) is highly dynamic and essential for viral entry into host CD4 + T cells 1 , 2 . Env is composed of three gp120 receptor binding subunits and three gp41 transmembrane subunits 2 . The fusion of viral and host membranes is initiated by receptor engagement of gp120 with human CD4, which induces conformational changes in Env that expose binding sites for coreceptors CCR5 or CXCR4 3,4 . Subsequent binding of these human coreceptors triggers insertion of the viral fusion peptide (FP) into the host membrane, eventually leading to viral genome entry, viral replication, and integration 5 . Due to the intrinsic instability of the HIV-1 Env, the SOSIP.664 modification was developed to stabilize soluble Env in its native-like state 6 , enabling structural characterization of Env in both closed and CD4-bound open states 7 – 12 . Comparative analyses of these states have revealed that CD4 binding triggers a cascade of structural rearrangements: burial of FP in a CD4-induced Env pocket, outward rotation of gp120, formation of a four-stranded bridging sheet and α0 helix, and displacement of V1V2 loops to expose the coreceptor binding site 10 – 12 . For clarity, we define the coreceptor binding site occluded closed conformational state as occluded closed, and the CD4-bound coreceptor binding site exposed conformational state as CD4-bound accessible open. Recently, cryo-EM and cryo-ET studies have elucidated stepwise CD4 engagement with Env, providing biological relevance to the structural intermediates of SOSIP-stabilized Env 13 , 14 . CD4 engagement begins with the binding of one CD4 molecule, resulting in an asymmetric open or closed conformational state of Env. Subsequent binding of a second or even a third CD4 molecule results in the asymmetric or symmetric opening of Env respectively, accompanied by a ~ 50 Å reduction in viral-envelope-to-host-membrane distance. Notably, cryo-ET reveals that three CD4-bound Env adopts a partially open state distinct from the fully open state typically observed by cryo-EM 11 , 14 . Additionally, occluded open states – exhibiting gp120 rotation but without V1V2 displacement, bridging sheet or α0 helix formation – have been captured by antibody binding and characterized by both cryo-ET and cryo-EM 10 , 15 – 17 . These occluded open states, alongside evidence that Env spontaneously samples open conformations without CD4 12,18–21 , suggest that CD4 engages Env in a pre-existing conformational continuum. To delineate the interplay between occluded closed, occluded open, and accessible open states, we characterized novel conformations of AMC008 Env from a clade B Tier-1B virus. AMC008 Env has been reported as highly conformationally heterogeneous; however, SOSIP.v4.2 stabilization (I535M, L543N, A316W, H66R, and SOSIP.664) enables expression of soluble, flexible AMC008 SOSIP.v4.2 Env (hereafter AMC008) 22 . We identified novel Env conformations by complexing AMC008 with antibody 3BC315 (targeting the gp41-gp41 interface) 23 , antibody b12 (targeting the CD4 binding site; CD4bs) 24 , CD4, or a combination of 3BC315 and b12. 3BC315 and b12 were chosen based on prior insights: b12 facilitated the first cryo-EM structure of occluded open Env 10 , while 3BC315 destabilizes Env via its interactions with the gp41-gp41 interface 25 . Cryo-EM structures of these complexes (resolutions ranging between 2.9–3.9 Å), alongside an occluded closed reference structure determined to a resolution of 2.9 Å, unveiled distinct conformational states including a base-relaxed state upon 3BC315 binding, unique occluded moderately open states upon b12 or CD4 binding, and an asymmetric occluded open state upon combined b12 and 3BC315 binding. Integrating these results with previous studies, we propose a classification framework to characterize Env conformations and a mechanism by which HIV-1 Env transitions from occluded closed to accessible CD4-bound fully open en-route to membrane fusion. We observed enhanced 3BC315 binding occupancy in the presence of b12, and discovered that when engaging Env, antibodies 3BC315 and b12 interact with each other directly. Moreover, we decipher Env allosteric mechanisms underlying the cooperative accommodation of 3BC315 and b12, resulting in higher occupancy and increased neutralization. Our findings refine the mechanistic understanding of HIV-1 Env dynamics and provide a structural roadmap for targeting dynamic Env states in more potent designs of vaccines and immunotherapeutics. Results 3BC315 binds a quaternary epitope 3BC315 binding to Env was previously reported to dissociate Env trimers 25 . To stabilize the 3BC315-AMC008 complex, we added VRC01 targeting the CD4bs and PGT121 targeting the V3-glycan epitope 26 , 27 . We determined a cryo-EM structure of the resulting AMC008-PGT121-VRC01-3BC315 complex at 3.2 Å (Fig. 1 a). In the data set, we observed two classes having complexed either one copy of 3BC315 or none at all, whereas both classes exhibited full VRC01 and PGT121 occupancy (Extended Data Fig. 5 d). This structure reveals atomic details of the 3BC315 epitope-paratope interface. To assess conformational changes in 3BC315 upon Env engagement, we superimposed the Env-bound 3BC315 Fab with its unliganded crystal structure (Cα RMSD = 0.6 Å; Extended Data Fig. 2 c) 25 . While the two structures are highly similar, key differences include the side chain reorientation of F56 CDRH2 to accommodate the N88 gp120 glycan and the side chain reorientation of R27 CDRL1 to hydrogen-bond with N543 gp41 (Extended Data Fig. 2 d). 3BC315 binds predominantly to one gp41 subunit but exhibits additional interactions with gp120 (same protomer) and adjacent gp41. The epitope is bordered by N88 gp120 and N625 gp41 glycans (Extended Data Fig. 1 a). The N88 gp120 glycan is axially flipped 180° and tilted 70° toward the Env apex to accommodate 3BC315 (Extended Data Fig. 1 b). Both 3BC315 heavy and light chains interact extensively with AMC008 with buried surface areas of 915 Å 2 and 503 Å 2 on the epitope, respectively (Extended Data Fig. 1 a). CDRH2, CDRH3, CDRL1 and CDRL3 of 3BC315 form polar interactions with the epitope to stabilize the binding of 3BC315. (Extended Data Fig. 1 c). A hydrophobic groove formed by six aromatic residues (CDRL1 Y30 and F32; CDRL3 Y91, Y94, Y100 E , and F100 I ) accommodates an Env α-helix spanning the fusion peptide proximal region (FPPR) and the N-terminal region of heptad repeat 1 (HR1N) (Extended Data Fig. 1 d). Additionally, the 3BC315 CDRH3 loop inserts into a cavity at the gp120-gp41-gp41 interface, positioning its tip at the hydrophobic gp120-gp41 interface (Extended Data Fig. 1 e). 3BC315-bound base-relaxed AMC008 The AMC008-PGT121-VRC01-3BC315 structure reveals remodeling of the Env base as compared to occluded closed Envs 9 . To investigate this base remodeling, we solved a 2.9 Å cryo-EM structure of occluded closed AMC008 by complexing AMC008 with VRC01 Fab and 35O22 Fab (targeting the gp120/gp41 interface) (Extended Data Fig. 2 a, 6 a) 28 . Superimposition of the AMC008-PGT121-VRC01-3BC315 and occluded closed AMC008-VRC01-35O22 structures demonstrates dramatic displacements by more than 5 Å in epitope-associated structural elements upon 3BC315 binding (Fig. 1 b). A hinge region comprised of residues L523 gp41 -S534 gp41 undergoes rigid-body movement, coupled with inward movement of FP toward the Env core and outward movement of HR1N (Fig. 1 b,c). Within the 3BC315-bound protomer, FP is embedded in a cavity formed by the non-helical α0 loop, the β0 strand, the α0-β0 connecting loop, and remodeled HR1N (Fig. 1 d). Furthermore, we aligned our 3BC315-bound gp41 to other gp41 remodeled Envs in the literature 10 , 11 , 29 – 32 . The 3BC315-bound gp41 resembles gp41s of T/F100-8ANC195 or CAP256.wk34.c80 SOSIP.RnS2, whereas, similar to our AMC008-PGT121-VRC01-3BC315, the FP burial occurs despite retention of occluded closed gp120 conformation (Fig. 1 e,f). Notably, FP burial is a hallmark of Env opening 10 . In occluded partially open CNE40-HmAb64 and accessible partially open BG505-8ANC195-17b-CD4, FPs adopt conformations similar to 3BC315-bound gp41 but with FPs less extended to avoid clashes with the C-terminal region of HR1 (HR1C). In contrast, in occluded fully open B41-b12 and CD4-bound accessible fully open B41-17b-CD4, FP burial persists but with further retraction to accommodate HR1C (Fig. 1 f). Based on this data we conclude that FP conformation in 3BC315-bound AMC008 gp41 is equivalent to gp41 conformations in T/F100-8ANC195 and CAP256.wk34.c80 SOSIP.RnS2, and this conformation most resembles partially open states. Thus, we define this FP buried closed conformational state as the base-relaxed state. In contrast, FP of BG505-PGT122-VRC34.01 sits in an opposite orientation away from the Env core directed by antibody VRC34.01 (Fig. 1 f). Taken together, these results underscore FP conformational plasticity across closed, partially open, and fully open Env states. To exclude potential confounding effects of VRC01/PGT121 on our observed base-relaxation, we determined cryo-EM structures of AMC008 bound solely to 3BC315. Unlike the AMC008-PGT121-VRC01-3BC315 complex that exhibited no more than single 3BC315 occupancy, the 3BC315-only dataset resolved two 3D classes: AMC008 bound to one (3.6 Å) or two (3.5 Å) 3BC315 Fabs (Extended Data Fig. 2 b, 5 d, 6 b). More importantly, all 3BC315-bound protomers adopt the base-relaxed conformation, while unbound protomers retain the closed conformation (Extended Data Fig. 2 e), confirming that remodeling is driven by 3BC315 engagement rather than auxiliary antibodies. b12-bound moderately open AMC008 We next determined the cryo-EM structure of AMC008 in complex with b12 at a resolution of 3.9 Å (Fig. 2 a and Extended Data Fig. 5 b). Surprisingly, the degree of gp120 opening observed in the AMC008-b12 structure is significantly reduced compared to the previously resolved B41-b12 structure 10 . To quantify gp120 openness in AMC008-b12, we measured interprotomer distances for representative Cα residues at the V3 base (H330 gp120 ), V1V2 base (P124 gp120 ), and CD4bs (D368 gp120 ). These measurements were compared across closed (AMC008-VRC01-35O22), partially open (CNE40-HmAb64, BG505-8ANC195-17b-CD4), and fully open (B41-b12, B41-17b-CD4) Env structures (Extended Data Fig. 3 a,b,d-g) 10 , 11 , 32 . The AMC008-b12 openness is intermediate between closed and partially open states, leading us to define this as a moderately open state. Subsequent alignment of gp41 across these structures reveals distinct HR1C conformations: in partially and fully open states, the HR1C helix extends by at least one additional N-terminal turn and rotates in divergent directions relative to the closed state. While AMC008-b12 also exhibits N-terminal helical extension of HR1C, its orientation remains similar to a closed state (Fig. 2 e). Integrating gp120 openness, gp41 conformation, and CD4-binding status, we categorized Env states as follows: A (closed), A BR (base-relaxed closed), B (moderately open), C (partially open), C’ (CD4-bound partially open), D (open) and D’ (CD4-bound open). Notably, significant displacement was observed at the β \(\:\stackrel{-}{4}\) /β26 strands of the gp120 N- and C-termini, which insert into the gp41 ‘four-helix collar’ – identifying this region as a pivotal point for gp120 rotation (Fig. 2 b). Displacements in the V1, V2, and V4 loops likely reflect inherent loop flexibility and isolate-specific sequence differences. Docking b12 Fab onto closed AMC008 reveals steric clashes with N197 and N301 glycans (Fig. 2 c). Upon b12 binding, the N197 glycan reorients to avoid clashes, accompanied by limited gp120 opening. This limited opening is sufficient for b12 accommodation, suggesting that SOSIP.v4.2-stabilized AMC008 samples open states less readily than B41 SOSIP (Fig. 2 d). The gp41 conformation in AMC008-b12 (state B) resembles gp41 of the closed state A, with solvent-exposed FP and bent FPPR/HR1N helix. However, HR1C in AMC008-b12 exhibits a 4.4° axial rotation toward the central symmetry axis and HR1N in AMC008-b12 is better resolved compared to state A (Fig. 2 f). Trimeric gp41 assemblies align closely between CD4-bound and unbound states for both partially open (C/C’) and fully open (D/D’) conformations (Fig. 2 g,h). Notably, while HR1C conformations in states B, C, and D appear distinct when aligned by protomer, their overall helical bundle compactness is similar when aligning full trimeric assemblies (Fig. 2 i). CD4-bound moderately open AMC008 Observing distinct conformational differences between occluded open AMC008-b12 and B41-b12 complexes, we hypothesized that CD4 binding to AMC008 would induce an alternative conformation. We therefore determined the cryo-EM structure of the AMC008-CD4 complex at a resolution of 2.9 Å (Fig. 3 a and Extended Data Fig. 5 e). Interprotomer distance measurements revealed that CD4-bound AMC008 exhibits intermediate gp120 openness, positioned between closed and partially open Envs but less open than AMC008-b12 (Extended Data Fig. 3 a-g). CD4 binding destabilized interprotomer contacts at the trimer apex, rendering the V1V2V3 region more flexible and disordered (Fig. 3 a). Local classification of the apex (Extended Data Fig. 5 e) yielded a 3.6 Å map showing that AMC008-CD4 gp120 retains occluded Env-like features: disordered but non-displaced V1V2 loops maintain the coreceptor binding site occluded despite partly disordering of V3, with no formation of the α0 helix or 4-stranded bridging sheet (Fig. 3 b). This structural arrangement explains the finding that CD4 binding to AMC008 SOSIP.v4.2 only marginally enhances binding of 17b, an antibody targeting the coreceptor binding site 22 . Additionally, two previous reports identified single CD4-bound Envs in closed conformation with occluded coreceptor binding sites (state A’) 13 , 33 . As AMC008-CD4 retains gp41 assembly similarity to AMC008-b12 despite differing gp120 openness, we classify it as state B’ (CD4-bound occluded moderately open) under our established criteria (Fig. 3 c). The binding of CD4 does not result in the formation of a 4-stranded bridging sheet in AMC008-CD4, however, the coordinates of β20/β21 align well with other CD4-bound Env gp120s to form the essential contacts with the CD4 F43 residue, indicating the remodeling of β20/β21 is presumably the first step of CD4 engagement (Fig. 3 d). This aligns with prior studies showing that restricting β20/β21 mobility via an interdomain 113C-429C disulfide bond impairs CD4 binding, underscoring the importance of β20/β21 remodeling for CD4 engagement 34 . Comparisons of the closed, the CD4-bound, and the b12-bound AMC008 structures reveal that W571 gp41 transitions from deeply buried between α0–1 and β \(\:\stackrel{-}{4}\) (state A/A’) to exiting this hydrophobic pocket in states B/B’ as a consequence of HR1C helix bundle compaction (Fig. 3 e). Based on this analysis, we find the HIV-1 Env trimer adopts distinct conformational states characterized by progressive gp120 opening and gp41 rearrangements. The occluded closed state A features tightly packed gp120 subunits at the trimer apex, solvent-exposed FP, bent FPPR/HR1N helices, and a non-extended HR1C bundle. A variant of this state, base-relaxed closed state A BR , retains most features of state A but adopts a gp41 base conformation resembling the partially open state C. In the occluded moderately open state B, gp120s rotate outward moderately, while gp41 retains state A-like features except for an extended and compacted HR1C bundle. Further opening defines the occluded partially open state C, where gp120s rotate more prominently, FP buries as a helical segment, and FPPR/HR1N adopts bent or straight conformations. The occluded fully open state D exhibits maximal gp120 rotation, a compact HR1C bundle, and FP repositioned into a shorter loop to avoid clashes. CD4 binding induces state-specific changes: it leaves state A’ unaltered relative to state A, destabilizes the V1V2V3 apex in state B’, displaces V1V2 while forming the α0 helix and the 4-stranded bridging sheet to expose the coreceptor binding site in states C’/D’ (Fig. 3 f). 3BC315 and b12-bound asymmetric open AMC008 Having observed alternative conformational changes upon binding either b12 or 3BC315, we rationalized that complexing AMC008 with both antibodies might lead to further structural rearrangements. We determined a cryo-EM structure of the AMC008-b12-3BC315 complex at a resolution of 3.7 Å (Fig. 4 a and Extended Data Fig. 5 c). Unexpectedly, with the presence of b12, the binding occupancy of 3BC315 increased from one or two in our base-relaxed structures to three. Additionally, unlike the limited degree of gp120 opening observed in the AMC008-b12 complex, gp120s of the AMC008-b12-3BC315 complex are more open but in an asymmetric manner. Heavy chain framework region (C H1 domain) of one b12 Fab interacts with light chain framework region (C L domain) of one 3BC315 Fab indicating a mechanism to enhance binding of the two antibodies by cooperativity (Fig. 4 a) 35 , 36 . Despite the global conformational opening of AMC008 and increased 3BC315 binding stoichiometry compared to our solely 3BC315 bound structures, there are only subtle local conformational changes in FP N-terminus and FPPR of the 3BC315 epitope. However, HR1C in each protomer rotates, becomes more compact, and forms additional helical turns, forcing FP to adopt a less extended conformation to prevent clashes with the rotated HR1C compared to the base-relaxed A BR state (Fig. 4 b). We next compared AMC008-b12-3BC315 to other published asymmetric open Env structures (Extended data Fig. 3 h-p) 12 , 13 , 17 , 37 , 38 . This gp120 conformation of AMC008-3BC315-b12 is similar to two published CD4-bound asymmetric open Envs, HT2-CD4 and BG505-E51-CD4 class II, evaluated by measuring the interprotomer residue distances of gp120 in these structures and global alignments (Fig. 4 c and Extended data Fig. 3 h-j). Alignment of AMC008-b12-3BC315 to HT2-CD4 and BG505-E51-CD4 class II by protomer reveals that they are similar at each position regardless of CD4 binding. Despite these similarities, gp120s of AMC008-b12-3BC315 lack the features of a CD4-bound accessible open conformation (Fig. 4 d). We further compared gp41 conformations of AMC008-b12-3BC315 by aligning each protomer to the symmetric open reference structures (Fig. 4 e). We observed that gp41 of each protomer can be matched with states C, or D, specifically, protomer 1 aligns well with state C while protomers 2 and 3 align better with state D. Therefore, we denote the b12 and 3BC315 bound AMC008 as state CDD. Furthermore, protomers of the currently existing asymmetric open Envs can be categorized using the same approach; thus, our state classification framework for symmetric Envs can be branched out to properly denote asymmetric Envs (Fig. 4 e and Extended Data Table 1). From these models of asymmetric or symmetric Envs, we further differentiated protomeric states C and D based on the coordination of W571 gp41 : the side chain of W571 gp41 is above the α0-β0 connecting loop in state C; but is beneath the loop and is buried in a hydrophobic pocket formed by F53 gp120 , P79 gp120 , C218 gp120 , and C247 gp120 in state D (Fig. 4 f). Together with states A/B/B’, we identify W571 gp41 to be a crucial residue that changes position corresponding to the conformational state of HIV-1 Env, consistent with previous structural and functional studies of Env W571 gp41 12,39,40 . To study the biological relevance of the states C’ and D’, we docked Env models of states C’/D’ into a cryo-ET density map of three CD4-bound Env, and then aligned gp120-CD4-CCR5 and AlphaFold-predicted full-length CD4 models to the docked Envs (Fig. 4 g) 14 , 41 , 42 . Using three copies of full-length CD4 and gp120-CD4-CCR5, we approximated the membrane surfaces of CCR5 and CD4. The approximated membrane surface of CD4 in state C’ matches the membrane density, while state D’ does not, consistent with the observation that Env in complex with CD4 imaged by cryo-ET adopts a partially open state 14 . We discovered that the membrane surfaces for CCR5 and CD4 do not positionally match in state C’ but appear to be much closer in D’. Based on this analysis, we predict that the virus-to-host membrane distance for state D’ will likely be ~ 110 Å, while the distance is measured to be ~ 140 Å in state C’ by cryo-ET 14 . Therefore, we expect states A’, B’, and C’ all to be early states which do not engage the co-receptor, while state D’ is likely primed to engage with the coreceptor and followed by membrane insertion of FP and dissociation of gp120. Structural and functional cooperativity of b12 and 3BC315 Cryo-EM analysis of the AMC008-3BC315 complex revealed two distinct populations: 48.2% of particles were bound to a single 3BC315 Fab, while 51.8% accommodated two Fabs per trimer (Extended Data Fig. 6 b). Strikingly, in the AMC008-PGT121-VRC01-3BC315 complex, 67.4% of particles retained one 3BC315 Fab, with the remaining 32.6% lacked 3BC315 entirely (Extended Data Fig. 5 d). In contrast, the AMC008-b12-3BC315 complex exhibited uniform occupancy, with three 3BC315 Fabs bound per trimer. These findings suggest that VRC01 and PGT121 stabilize Env in the occluded closed state (State A), which allosterically impedes 3BC315 binding, whereas b12 promotes a more open state (State B), enabling full 3BC315 occupancy. Moreover, as described above, simultaneous binding of b12 and 3BC315 facilitates stabilization of the complex through multiple hydrogen bonds formed between their constant framework regions (Fig. 4 a). To validate the cooperative binding of b12 and 3BC315 to Env, we performed mass photometry experiments (Fig. 5 a and Extended Data Fig. 4 ). The molecular mass of trimeric native-like AMC008 was estimated at approximately 338 kDa. Upon binding excess 3BC315 Fab, the mass increased by 114 kDa (consistent with ~ 2 Fabs), whereas excess b12 Fab or VRC01 Fab increased the mass by 170 kDa or 164 kDa, respectively (~ 3 Fabs each). Consistent with our cryo-EM results, adding excess 3BC315 Fab to the AMC008-b12 complex induced an additional 162 kDa increase (~ 3 Fabs). A distinct peak corresponding to AMC008-b12 without 3BC315 binding was observed, suggesting an all-or-none binding stoichiometry for 3BC315 Fab in the presence of b12. This implies that, in the context of b12, initial 3BC315 Fab binding markedly enhances affinity for the remaining two sites, indicative of positive cooperativity. In contrast, adding excess 3BC315 Fab to the AMC008-VRC01 complex yielded only a 66 kDa increase (~ 1 Fab), reflecting negative cooperativity. These results align with our cryo-EM data, confirming that b12 facilitates 3BC315 binding to the AMC008 trimer, while VRC01 antagonizes it. b12 is a CD4bs bNAb that exhibits limited binding and neutralization to tier 2 or higher HIV isolates, likely due to restricted conformational flexibility in these Env trimers. Our structures of AMC008-b12 and AMC008-b12-3BC315 reveal that while b12 binds to moderately open Env, the combination of both b12 and 3BC315 leads to an even more open Env conformation. We hypothesized that this cooperative binding, observed structurally, would enhance neutralization compared to either antibody alone, even against resistant isolates. To test this, we performed pseudovirus neutralization assays. 3BC315 neutralized all tested strains, whereas b12 only showed activity against SF162 and BaL, as previously reported and confirmed by our data (Fig. 5 b,c) 23 , 24 . When the two antibodies were used in combination (Fig. 5 d-h), our neutralization assays showed a synergistic increase in neutralization activity, suggesting enhanced engagement of Env on the viral envelope. This neutralization synergy was pronounced for strains that could be neutralized by either b12 or 3BC315 – SF162 and BaL (Fig. 2 e,f). Interestingly, even for strains completely resistant to b12 neutralization – BG505_T332N and TRO11, the presence of b12 still enhanced the neutralization activity when in combination with 3BC315, though less significantly than against strains that can be neutralized by b12 or 3BC315 (Fig. 5 d,g). These findings demonstrate that structural cooperativity between b12 and 3BC315 correlates with functional enhancement, with implications for antibody combination therapies. Discussion In this study, we solved cryo-EM structures of base-relaxed asymmetric AMC008 with 3BC315 at atomic resolution, revealing conformational changes that resemble open Env conformations at the gp41 binding interface, namely FP, FPPR, and HR1N. Additionally, we solved structures of AMC008 bound with CD4 or b12 alone and with 3BC315. In our b12-bound or CD4-bound AMC008 structures, we found a new Env conformational intermediate, coined moderately open conformation, between the closed and partially open Env conformations with a moderately rotated gp120 and an N-terminally extended and more compacted HR1C. On the other hand, 3BC315 was found to induce a local gp41 conformational change without changing the conformation of gp120. Our structure of b12 and 3BC315 in complex with AMC008 revealed an alternative asymmetric occluded open Env conformation that resembles the two CD4-bound and three CD4-bound asymmetric open Env structures 12 , 13 . We observe full occupancy (three copies of b12 and three copies of 3BC315 bound) in this asymmetric occluded open state, and we observe hydrogen-bonding ability between b12 C H1 and 3BC315 C L constant IgG domains. These structural arrangements suggest a cooperativity mechanism between the two antibodies that combines framework antibody stacking ability as well as antibody manipulation of HIV-1 Env allostery to enhance combined antibody affinities to their respective epitopes. We confirmed our structural observations of antibody cooperativity using mass photometry and pseudovirus neutralization assays. Utilizing the effect of cooperativity between a combination of antibodies could be a potential advantage when developing prophylactic or therapeutic antibody cocktails leading to overall increased potency and breadth. Furthermore, 3BC315 exhibits the unique property of capturing an alternative gp41 conformation in a closed Env (AAA BR ) that potentially shifts the conformational equilibrium of HIV-1 Env towards more open and immunogenic states. Developing vaccination strategies to elicit these types of broadly neutralizing antibodies could be another viable prophylactic avenue due to their ability to recover the increased neutralization susceptibility of Env toward neutralizing antibodies that bind open conformations. Until now, the conformational flexibility of Env has been challenging to comprehend, partially due to a lack of a systematic approach towards classifying intermediates from closed to open conformations, especially for pre-CD4 engaged conformations. Recent cryo-EM and cryo-ET studies have shown that asymmetric Env conformations are biologically relevant and exist in the context of one, two, or three CD4-bound Envs 13 , 14 . In addition, multiple smFRET studies have shown that unliganded Env on a native virion also spontaneously sample similar conformations that are enriched after CD4 engagement 19 , 43 , 44 . Furthermore, spontaneous opening of HIV-1 Env was observed in solution DEER spectroscopy, HDX-MS, and SAXS 18 , 20 , 21 . Therefore, we propose a model of the conformational trajectory of HIV-1 Env (Fig. 6 ) that encompasses the trimeric conformational states that include closed (A), base-relaxed closed (A BR ), moderately open (B), partially open (C), asymmetrically open (BCD, CCD, and CDD) and finally, fully open (D) states by categorizing each protomer of Env. In our proposed model, Env exhibits a natural ability to sample through stepwise opening from states A, B, C, and finally, D. Note that this conformational equilibrium is heavily skewed towards A for tier 2 and 3 isolates, while tier 1 HIV-1 isolates, such as B41, seem to sample these conformations equally as shown recently shown by DEER spectroscopy 18 . Although assigned as a closed state, local base-relaxation of FP/FPPR/HR1N may occur rarely and be captured by antibody binding 30 , 31 . Our AMC008-b12 complex is the first symmetric state B Env, and it is a crucial intermediate to delineate the transition from state A to state C. For Env to transition from closed to open conformations, it must assume state B to avoid the steric clash occurring at the C and D strands of the V2 loop (Movies 1 and 2). In line with our observations, SAXS studies suggest the existence of an early intermediate conformation of HIV-1 Env that involves increased flexibility around the apex loop contacts 20 and smFRET studies further show that binding of one CD4 leads to the adjacent Env protomer sampling an intermediate conformational state between the closed state and CD4-bound open state 44 . This conformational state in the protomer adjacent to the CD4-bound protomer most likely corresponds to state B of the present work. Our models of AMC008-b12 (B) and AMC008-CD4 (B’) provide the first structural evidence for this intermediate conformation. CD4 can engage Env at each state; however, the coreceptor binding site is only accessible in CD4-bound states C’ and D’, but not in CD4-bound A’ and B’. By CD4 and CCR5 membrane surface measurements, we tentatively conclude that the coreceptor engages Env in state D’; and states A’, B’, and C’ are early intermediates of CD4 engagement (Fig. 4 g). Thus, we hypothesize at least one protomer of Env in state D’ is required for Env transition to postfusion. Our present model for the possible conformational states provides a framework of the conformational plasticity of HIV-1 Env and provides a template for therapeutic development and immunogen design strategies. Methods DNA constructs Constructs for transient expression of antibodies 35O22, VRC01, b12, and PGT121 in mammalian cells were obtained from the Kulp group at the Wistar Institute (Philadelphia, PA). Construct for expression of antibody 3BC315 in mammalian cells was designed in-house and synthesized by a commercial vendor (Genscript). The full-length BG505 plasmid was used to make point mutations at T332N for pseudotype virus production. Plasmids expressing HIV backbone ΔEnv (pSG3), SF162, BAL, TR011, and murine leukemia virus (MLV) control envelope were obtained from the NIH AIDS Reagents Program. AMC008 SOSIP.v4.2 expression and purification AMC008 SOSIP.v4.2 Env and furin were transiently co-transfected in Expi293F cells (Gibco) at a cell density of 2.5x10 6 cells/mL. The supernatant of the cell culture was harvested five days after transfection, or once cell viability was below 50%, by centrifugation at 6000g for 30 minutes, followed by 0.2 μm filtration and the addition of 1 mM PMSF. 0.5 mL lectin resin (Vector Laboratories) was added to the supernatant and incubated overnight. The overnight incubated supernatant was loaded and flowed through a gravity column (Bio-Rad), washed with 10 column volumes (CV) of PBS, and eluted with 5 CV of elution buffer (1M Methyl α-Mannopyranoside in PBS). The eluent was desalted by a PD-10 desalting column (Bio-Rad) and concentrated to 1 mL by centrifugal filter with a 100 kDa molecular weight cutoff (Millipore). The lectin-purified and desalted AMC008 Env protein was a mixture of AMC008 aggregate, trimer, and monomer. The mixture was purified by SEC using a 10/300 Superose 6 Increase column (Cytiva). Fractions corresponding to the AMC008 Env trimer were collected, concentrated using a centrifugal filter with a 100 kDa molecular weight cutoff to 1g/L, snap-frozen, and stored at -80 °C for future usage. Antibody digestion and Fab purification The heavy chain and light chain of antibodies were transiently co-expressed in Expi293F cells with a ratio of 3:2. The supernatants of the cell cultures were harvested seven days after transfection or once cell viability was below 50% similarly to AMC008 Env. 1 mL Protein A resin (Cytiva) was added to the supernatant and incubated overnight. The overnight incubated supernatant was loaded onto a gravity column, washed with 10 CV of PBS, and eluted with 5 CV of elution buffer (20 mM sodium citrate, 150 mM sodium chloride, pH 2.5). Eluent was collected into a 50 mL centrifugal tube containing 5 mL of 1M pH 8 Tris to neutralize the elution buffer. Eluted antibodies were buffer-exchanged and concentrated to 5-10 mg/mL by centrifugal filtration with a 100 kDa molecular weight cutoff and stored at 4°C. The purified antibodies were diluted to around 1 g/L in digestion buffer (10 mM L-cysteine, 100 mM sodium acetate, 0.3 mM EDTA, pH 5.6). Papain (Millipore) was pre-incubated in digestion buffer at 37°C for 10 minutes. The activated papain was added to the antibody and incubated at 37°C overnight. Digestions were terminated by adding 3 mM iodoacetamide. The digested mixtures containing Fab, Fc, and undigested antibody were purified by Protein A resin. 1 mL of Protein A resin was added to the mixtures and loaded onto a gravity column and incubated for 10 minutes. Flow-through was collected after incubation, concentrated, and buffer exchanged to PBS in a centrifugal filter with a 10 kDa molecular weight cutoff (Millipore). 10 CV of PBS was added to wash the column. 5 CV of Protein A elution buffer was added to elute Fc and undigested antibody. Fabs were stored at 4 °C for future usage. Complexation and cryo-EM sample preparation Complexes were formed by incubating AMC008 Env with Fabs or CD4 in a 1:9 molar ratio in PBS for at least one hour at 4 °C. The mixture was purified by SEC using a 10/300 Superose 6 Increase column. The fractions corresponding to the complex were collected, combined, concentrated to 1 g/L, snap-frozen, and stored at -80 °C for future use. Complexes were thawed, diluted to around 0.1 g/L, and placed on ice for cryo-grid preparation. GF-1.2/1.3-3Au-45nm cryo-EM grids (Protochips) were glow-discharged for 30 seconds, and the glow-discharged grids were coated with graphene oxide (GO). Complexes were vitrified by applying 4 μL diluted sample to the GO-coated grid using the vitrobot Mark IV (FEI) at 4 °C and 100% humidity, followed by plunging into liquid ethane. Cryo-electron microscopy data collection and processing Cryo-EM data were collected with a 300 kV Titan Krios G3i microscope equipped with a K3 Summit Direct Electron Detector camera at 81,000x magnification, a nominal dose of 58 e-/Å2, and a defocus range from -0.5 μm to -2.5 μm. Movies were processed by RELION v3.1 45 using a standard cryo-EM data processing workflow. Workflow included motion correction, CTF estimation, LoG picking, particle extraction, 2D classification, manual selection of good 2D class averages, 3D classification and refinement, and Bayesian polishing. The polished particles were imported into CryoSPARC 46 for Non-uniform refinement. Consensus maps of AMC008-PGT121-VRC01-3BC315, AMC008-VRC01-35O22, AMC008-b12, AMC008-CD4, AMC008-3BC315, and AMC008-b12-3BC315 were generated with the workflow mentioned above (Extended Data Fig. 5, 6). In the AMC008-PGT121-VRC01-3BC315 dataset, we observed zero or one 3BC315 binding to the AMC008 SOSIP.v4.2 Env. We performed a classification focused on the 3BC315 binding site with three classes. Two classes that had 3BC315 binding were combined and reconstructed to the final AMC008-PGT121-VRC01-3BC315 map (Extended Data Fig. 5d). In the AMC008-CD4 dataset, we observed one to three CD4s binding to AMC008 SOSIP.v4.2. We simulated three density maps of one, two, and three CD4s binding to AMC008 SOSIP.v.4.2, and did a heterogeneous refinement with the simulated maps. The resulting 3D classes of AMC008-CD4 were all in the same conformational state despite the binding occupancy of CD4. We reconstructed the final map with particles of the three CD4-bound class and imposed C3 symmetry, as the three CD4-bound class was most populated and had the best view distribution. To investigate the V1V2V3 conformation of AMC008-CD4, we transferred particles of the three CD4-bound class back to Relion and performed a 3D classification focused on the Env apex with four classes. Two of the classes with density definition on Env apex were combined and imposed C3 symmetry to reconstruct a map with better density on Env apex. In the AMC008-VRC01-35O22 dataset, we observed exclusively three VRC01 Fabs binding to the Env, while one to three 35O22 Fabs bound to the AMC008 SOSIP.v4.2 Env when processing the cryo-EM data. Reconstructing each complex population with different 35O22 Fab binding occupancy, we found that the binding of 35O22 did not alter the AMC008 SOSIP.v4.2 Env conformation and only subtle differences were observed around the 35O22 epitope. After applying multiple data processing schemes, we found that combining populations with two or three 35O22 Fab binding occupancy and imposing C3 symmetry gave the best 3D reconstruction, considering both the global and local resolutions at the Env base (Extended Data Fig. 6a). In the AMC008-3BC315 dataset, we observed one or two 3BC315 binding to the AMC008 SOSIP.v4.2, and the ratio of the two populations was about 1:1. Reconstruction of the two classes showed dramatic conformational changes around the 3BC315 epitope. We performed a classification focused on the 3BC315 binding site with ten classes. We only included particles with high confidence in 3BC315 binding occupancy for the final round of reconstruction (Extended Data Fig. 6b). Model building and refinement Crystal structures of CD4 (PDB: 1WIO), VRC01 (PDB: 4LST), 35O22 (PDB: 4TOY), PGT121 (PDB: 4JY4), b12 (PDB: 1HZH), and 3BC315(PDB: 5CCK) were used as reference models. A reference model of AMC008 Env was generated by homology modeling using Modeler 47 . Reference models were rigid-body docked into the density maps in UCSF Chimera 48 and manually rebuilt in Coot 49 . N-linked glycans were added to the models according to the N-linked glycosylation consensus sequence and the density maps. The manually rebuilt models were further refined using Rosetta FastRelax 50 . Refined models were reinspected and adjusted in Coot and refined by Rosetta again. These steps were repeated until no significant improvement was observed. Model geometry was validated by MolProbity 51 , glycan conformation was validated by Privateer 52 , and model fit-to-map was validated by EMRinger 53 . Mass photometry Complexes for mass photometry were prepared by mixing AMC008 Env with Fabs in a 1:9 molar ratio in PBS and incubating on ice for 30 minutes. Samples were diluted in PBS to optimal concentrations right before data collection. Data were collected by a TwoMP mass photometer (Refeyn) in default settings for 60 seconds and were calibrated by a ladder comprising bovine serum albumin (66 kDa), β-amylase (224 kDa), and thyroglobulin (670 kDa). Raw data were analyzed using DiscoverMP software to generate mass histograms. Cell lines and transfections for neutralization assay HEK 293 T cells (ATCC) and TZM-bl cells (NIH AIDS Reagents Program) were maintained in DMEM (ThermoFisher) supplemented with 10% heat-inactivated fetal bovine serum (Atlas Biologicals). Pseudotype viruses were produced as previously described 54 . Briefly, HEK 293T cells were co-transfected with 4 µg of a plasmid encoding the desired Env protein and 8 µg of a plasmid expressing the HIV-1 backbone Δ Env (pSG3ΔEnv − NIH AIDS Reagents) using GeneJammer (Aglient). Forty-eight hours after transfection, cell supernatants were harvested, filtered using a 45 µm filter, aliquoted, stored at −80°C and titered. Neutralization assay Pseudotyped viruses were titered to yield 150,000 RLU after 48 h of infection with TZM-BL cells 54 . Monoclonal antibodies (MAbs) were serially diluted either individually or mixed together as combination at predetermined concentrations in 96-well plates followed by incubation with respective pseudotyped viruses before adding 10,000 TZM-BL cells (NIH AIDS Reagent Program) per well with dextran (ThermoFisher). Forty-eight hours post incubation, media was removed, and cells were lysed using BriteLite luciferase reagent (Revvity). Luminescence was then measured using the Synergy2 plate reader (BioTek Instruments). Statistical analysis All statistics and calculations were performed using GraphPad Prism 10.0. MAb titer was determined for 50% virus neutralization (ID50), values were computed and graphed with a nonlinear regression model of percentage neutralization vs log10 concentration of MAbs. Declarations Data availability The atomic models were deposited to PDB under accession codes 9NBT (AMC008-VRC01-35O22), 9NBY (AMC008-PGT121-VRC01-35022), 9NC0 (AMC008-b12), 9OAJ (AMC008-CD4), 9NC3 (AMC008-b12-3BC315), 9NC6 (AMC008-3BC315 (2x)) and 9NC8 (AMC008-3BC315 (1x)). The cryo-EM maps were deposited to EMDB under accession codes EMD-49236 (AMC008-VRC01-35O22), EMD-49238 (AMC008-PGT121-VRC01-35022), EMD-49239 (AMC008-b12), EMD-70287 (AMC008-CD4), EMD-49240 (AMC008-b12-3BC315), EMD-49241 (AMC008-3BC315 (2x)) and EMD-49242 (AMC008-3BC315 (1x)). Acknowledgements We thank Thomas Klose at the Purdue University Cryo-EM facility and Ruben Diaz Avalos at the La Jolla Institute for their assistance in sample screening and data collection. We thank Boyu Yin for his assistance in mass photometry experiments. This work was funded by W.W. Smith Charitable Foundation grant A2404 (to J.P.), NIH grant U19 AI166916 (to D.B.W and J.P). Funding sources were not involved in the design of this study, collection and analyses of data, decision to submit or preparation of the manuscript. Author contributions J.C. and J.P. conceived experiments. J.C. produced protein samples. J.D. collected cryo-EM data. J.C., J.D., Z.L., and J.P. processed the cryo-EM data. J.C. built atomic models. J.C. and Z.L. performed mass photometry experiments. S.G. and R.S. performed pseudovirus neutralization assays. J.C. analyzed and interpreted the data. J.C., Z.L., and J.P. wrote the manuscript draft. S.G. contributed to the manuscript draft. All authors reviewed and commented on the manuscript. J.P. and D.B.W. supervised the work and acquired funding. Competing interests D.B.W. notes several possible competing interests, which are managed by the Wistar General Council Office COI committee. These include consulting, BOD service, speaking, that can include in stock or monetary renumeration, and specific SRAs. Inovio Pharmaceuticals (BOD, consultant and SRA); AstraZeneca (speaker/consultant); Geneos (consultant & SRA); and possibly others that are managed by Wistar COI Committee. D.B.W. is a member of the International Society for Vaccines, AAI, ASGCT, AAAS, among other scientific societies. He also serves on NIH and NCI study sections and similar activities for other agencies. References Wyatt, R. & Sodroski, J. The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science 280 , 1884-8 (1998). Ward, A.B. & Wilson, I.A. The HIV-1 envelope glycoprotein structure: nailing down a moving target. Immunol Rev 275 , 21-32 (2017). Kwong, P.D. et al. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393 , 648-59 (1998). Berger, E.A., Murphy, P.M. & Farber, J.M. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu Rev Immunol 17 , 657-700 (1999). Blumenthal, R., Durell, S. & Viard, M. HIV entry and envelope glycoprotein-mediated fusion. J Biol Chem 287 , 40841-9 (2012). Sanders, R.W. et al. A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies. PLoS Pathog 9 , e1003618 (2013). Julien, J.P. et al. Crystal structure of a soluble cleaved HIV-1 envelope trimer. Science 342 , 1477-83 (2013). Lyumkis, D. et al. Cryo-EM structure of a fully glycosylated soluble cleaved HIV-1 envelope trimer. Science 342 , 1484-90 (2013). Kwon, Y.D. et al. Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env. Nat Struct Mol Biol 22 , 522-31 (2015). Ozorowski, G. et al. Open and closed structures reveal allostery and pliability in the HIV-1 envelope spike. Nature 547 , 360-363 (2017). Wang, H., Barnes, C.O., Yang, Z., Nussenzweig, M.C. & Bjorkman, P.J. Partially Open HIV-1 Envelope Structures Exhibit Conformational Changes Relevant for Coreceptor Binding and Fusion. Cell Host Microbe 24 , 579-592 e4 (2018). Yang, Z., Wang, H., Liu, A.Z., Gristick, H.B. & Bjorkman, P.J. Asymmetric opening of HIV-1 Env bound to CD4 and a coreceptor-mimicking antibody. Nat Struct Mol Biol 26 , 1167-1175 (2019). Dam, K.A., Fan, C., Yang, Z. & Bjorkman, P.J. Intermediate conformations of CD4-bound HIV-1 Env heterotrimers. Nature 623 , 1017-1025 (2023). Li, W. et al. HIV-1 Env trimers asymmetrically engage CD4 receptors in membranes. Nature 623 , 1026-1033 (2023). Liu, J., Bartesaghi, A., Borgnia, M.J., Sapiro, G. & Subramaniam, S. Molecular architecture of native HIV-1 gp120 trimers. Nature 455 , 109-13 (2008). Harris, A.K., Bartesaghi, A., Milne, J.L. & Subramaniam, S. HIV-1 envelope glycoprotein trimers display open quaternary conformation when bound to the gp41 membrane-proximal external-region-directed broadly neutralizing antibody Z13e1. J Virol 87 , 7191-6 (2013). Yang, Z. et al. Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer. Nat Commun 13 , 732 (2022). Stadtmueller, B.M. et al. DEER Spectroscopy Measurements Reveal Multiple Conformations of HIV-1 SOSIP Envelopes that Show Similarities with Envelopes on Native Virions. Immunity 49 , 235-246 e4 (2018). Lu, M. et al. Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET. Nature 568 , 415-419 (2019). Bennett, A.L. et al. Microsecond dynamics control the HIV-1 Envelope conformation. Sci Adv 10 , eadj0396 (2024). Hodge, E.A. et al. Structural dynamics reveal isolate-specific differences at neutralization epitopes on HIV Env. iScience 25 , 104449 (2022). de Taeye, S.W. et al. Immunogenicity of Stabilized HIV-1 Envelope Trimers with Reduced Exposure of Non-neutralizing Epitopes. Cell 163 , 1702-15 (2015). Klein, F. et al. Broad neutralization by a combination of antibodies recognizing the CD4 binding site and a new conformational epitope on the HIV-1 envelope protein. J Exp Med 209 , 1469-79 (2012). Burton, D.R. et al. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science 266 , 1024-7 (1994). Lee, J.H. et al. Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike. Nat Commun 6 , 8167 (2015). Zhou, T. et al. Structural basis for broad and potent neutralization of HIV-1 by antibody VRC01. Science 329 , 811-7 (2010). Julien, J.P. et al. Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans. PLoS Pathog 9 , e1003342 (2013). Huang, J. et al. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface. Nature 515 , 138-42 (2014). Kong, R. et al. Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody. Science 352 , 828-33 (2016). Ananthaswamy, N. et al. A sequestered fusion peptide in the structure of an HIV-1 transmitted founder envelope trimer. Nat Commun 10 , 873 (2019). Gorman, J. et al. Structure of Super-Potent Antibody CAP256-VRC26.25 in Complex with HIV-1 Envelope Reveals a Combined Mode of Trimer-Apex Recognition. Cell Rep 31 , 107488 (2020). Wang, S. et al. Human CD4-binding site antibody elicited by polyvalent DNA prime-protein boost vaccine neutralizes cross-clade tier-2-HIV strains. Nat Commun 15 , 4301 (2024). Liu, Q. et al. Quaternary contact in the initial interaction of CD4 with the HIV-1 envelope trimer. Nat Struct Mol Biol 24 , 370-378 (2017). Zhang, P. et al. Design of soluble HIV-1 envelope trimers free of covalent gp120-gp41 bonds with prevalent native-like conformation. Cell Rep 43 , 114518 (2024). Oyen, D. et al. Cryo-EM structure of P. falciparum circumsporozoite protein with a vaccine-elicited antibody is stabilized by somatically mutated inter-Fab contacts. Sci Adv 4 , eaau8529 (2018). Parzych, E.M. et al. DNA-delivered antibody cocktail exhibits improved pharmacokinetics and confers prophylactic protection against SARS-CoV-2. Nat Commun 13 , 5886 (2022). Jette, C.A. et al. Cryo-EM structures of HIV-1 trimer bound to CD4-mimetics BNM-III-170 and M48U1 adopt a CD4-bound open conformation. Nat Commun 12 , 1950 (2021). Wang, H. et al. Potent and broad HIV-1 neutralization in fusion peptide-primed SHIV-infected macaques. Cell 187 , 7214-7231 e23 (2024). Mo, H. et al. Conserved residues in the coiled-coil pocket of human immunodeficiency virus type 1 gp41 are essential for viral replication and interhelical interaction. Virology 329 , 319-27 (2004). Henderson, R. et al. Disruption of the HIV-1 Envelope allosteric network blocks CD4-induced rearrangements. Nat Commun 11 , 520 (2020). Shaik, M.M. et al. Structural basis of coreceptor recognition by HIV-1 envelope spike. Nature 565 , 318-323 (2019). Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596 , 583-589 (2021). Munro, J.B. et al. Conformational dynamics of single HIV-1 envelope trimers on the surface of native virions. Science 346 , 759-63 (2014). Ma, X. et al. HIV-1 Env trimer opens through an asymmetric intermediate in which individual protomers adopt distinct conformations. Elife 7 (2018). Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7 , e42166 (2018). Punjani, A., Rubinstein, J.L., Fleet, D.J. & Brubaker, M.A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14 , 290-296 (2017). Šali, A. & Blundell, T.L. Comparative Protein Modelling by Satisfaction of Spatial Restraints. Journal of Molecular Biology 234 , 779-815 (1993). Meng, E.C. et al. UCSF ChimeraX: Tools for structure building and analysis. Protein Science 32 , e4792 (2023). Emsley, P. & Cowtan, K. Coot: Model-building tools for molecular graphics. Acta Crystallographica Section D: Biological Crystallography 60 , 2126-2132 (2004). Tyka, M.D. et al. Alternate states of proteins revealed by detailed energy landscape mapping. J Mol Biol 405 , 607-18 (2011). Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallographica Section D 66 , 12-21 (2010). Agirre, J. et al. Privateer: software for the conformational validation of carbohydrate structures. Nature Structural & Molecular Biology 22 , 833-834 (2015). Barad, B.A. et al. EMRinger: side chain–directed model and map validation for 3D cryo-electron microscopy. Nature Methods 12 , 943-946 (2015). Sarzotti-Kelsoe, M. et al. Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1. J Immunol Methods 409 , 131-46 (2014). Additional Declarations There is NO Competing Interest. Supplementary Files ExtendedDataFigureandtableandmovielegends.docx movie1V2clash.mp4 movie1_V2_clash.mp4 movie2V2noclash.mp4 movie2_V2_noclash.mp4 Cite Share Download PDF Status: Published Journal Publication published 24 Feb, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6890430","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":479157685,"identity":"1c8461e7-5c7e-4d1f-b3ee-f47d9924abcd","order_by":0,"name":"Jesper Pallesen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYBACxgYGxgMPQCz2BoYDxGphOJAAYvEcAGpJINImiBYJEEmMFubZzQ8OJFTUyZlLvjE8+POHXT4De+/jF3gdNueYwYGEM4eNLWfnGBzmSUi2bOA5bmaBV8uMBIMDiW0HEjfczt1wmCGB2YBBIo3NAL+W9A8HEv/V1W+4eXbDwR8J9cRoyQHa0sCcYHCDd8MBnoTDIC3MD/D75UzBgYRjhw03nMn/cJgn7bgBG88xNnw6GAxnt2988KGmTt7g+LHkjz9sqg342duYP+DVMgNdBGgFmwQ+LfLYZPHbMgpGwSgYBSMOAADgoVKKZ+QcxwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-3270-1587","institution":"The Wistar Institute","correspondingAuthor":true,"prefix":"","firstName":"Jesper","middleName":"","lastName":"Pallesen","suffix":""},{"id":479157686,"identity":"108eba2f-46d5-4019-8c54-08f56b348926","order_by":1,"name":"Jiayan Cui","email":"","orcid":"","institution":"The Wistar Institute","correspondingAuthor":false,"prefix":"","firstName":"Jiayan","middleName":"","lastName":"Cui","suffix":""},{"id":479157687,"identity":"b8e6baf7-d14e-4d12-8649-c10dde3c2124","order_by":2,"name":"Zi Jie Lin","email":"","orcid":"","institution":"The Wistar Institute","correspondingAuthor":false,"prefix":"","firstName":"Zi","middleName":"Jie","lastName":"Lin","suffix":""},{"id":479157688,"identity":"ba67a7a8-a0be-40bf-9ffc-43a3956c6709","order_by":3,"name":"Sukanya Ghosh","email":"","orcid":"","institution":"The Wistar Institute","correspondingAuthor":false,"prefix":"","firstName":"Sukanya","middleName":"","lastName":"Ghosh","suffix":""},{"id":479157689,"identity":"8e3be2a1-cde9-4641-a7aa-4ece2d5f0d2b","order_by":4,"name":"Jianqiu Du","email":"","orcid":"","institution":"The Wistar Institute","correspondingAuthor":false,"prefix":"","firstName":"Jianqiu","middleName":"","lastName":"Du","suffix":""},{"id":479157690,"identity":"c75695cf-1eee-48ac-a0ea-27e4314a8c3d","order_by":5,"name":"Roopak Sadeesh","email":"","orcid":"","institution":"The Wistar Institute","correspondingAuthor":false,"prefix":"","firstName":"Roopak","middleName":"","lastName":"Sadeesh","suffix":""},{"id":479157691,"identity":"d0ab2bfc-1de6-4ecc-87bf-69455dd3c3aa","order_by":6,"name":"David Weiner","email":"","orcid":"https://orcid.org/0000-0002-2232-8512","institution":"The Wistar Institute","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Weiner","suffix":""}],"badges":[],"createdAt":"2025-06-13 19:20:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6890430/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6890430/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-026-69921-z","type":"published","date":"2026-02-24T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86301956,"identity":"eba88d52-d4b7-404f-a554-ff7623cc1e84","added_by":"auto","created_at":"2025-07-09 06:31:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2587970,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBase-relaxation of AMC008 SOSIP.v4.2. a\u003c/strong\u003e 3.2 Å cryo-EM density map (left and middle, top and side views) and atomic model (right) of AMC008 in complex with PGT121, VRC01 and 3BC315 Fabs. \u003cstrong\u003eb\u003c/strong\u003e Left: Cα RMSD of 3BC315-bound AMC008 in comparison to closed AMC008. Right: a close-up view of the 3BC315 binding epitope. \u003cstrong\u003ec\u003c/strong\u003e Comparison of FP, FPPR, and HR1N conformation with (orange) and without (gray) 3BC315 binding. C indicates C-termini, N indicates N-termini. \u003cstrong\u003ed \u003c/strong\u003eConformation of FP upon 3BC315 binding with surface representation of AMC008. The surfaces of AMC008 are colored by hydrophobicity. \u003cstrong\u003ee\u003c/strong\u003e Comparison of gp41 conformations in AMC008-PCT121-VRC01-3BC315, T/F100-8ANC195, and CAP256.wk34.c80 SOSIP.Rn32 Env structures. \u003cstrong\u003ef\u003c/strong\u003e Comparison of FP conformations in Env structures.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6890430/v1/2819a6acbd745137fc518786.png"},{"id":86302384,"identity":"d9a349ff-d6ad-4c1b-93fb-0d1ee7699752","added_by":"auto","created_at":"2025-07-09 06:39:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2570832,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRemodeling of AMC008 SOSIP.v4.2 upon b12 binding. a \u003c/strong\u003e3.9 Å cryo-EM density map (left and middle, top and side views) and atomic model (right) of AMC008 in complex with b12 Fab. \u003cstrong\u003eb\u003c/strong\u003e Left: superposition of the protomers of b12-bound B41 and AMC008. Right: Cα RMSD of b12-bound AMC008 protomer in comparison to b12-bound B41 protomer. \u003cstrong\u003ec\u003c/strong\u003e b12 cannot bind AMC008 in closed conformation due to clashes of N197 and N301 glycans with b12. Clashes are indicated by red * marks. \u003cstrong\u003ed\u003c/strong\u003e Binding of b12 requires minimum openness of AMC008 gp120 to accommodate N197 and N301 glycans. \u003cstrong\u003ee\u003c/strong\u003e Comparison of the HR1C conformations in \u003cstrong\u003eExtended Data Fig. 2a-f\u003c/strong\u003e. \u003cstrong\u003ef\u003c/strong\u003e-\u003cstrong\u003eh\u003c/strong\u003eSuperposition of gp41s in AMC008-VRC01-35O22 and AMC008-b12 (\u003cstrong\u003ef\u003c/strong\u003e), CNE40-HmAb64 and BG505-8ANC195-17b-CD4 (\u003cstrong\u003eg\u003c/strong\u003e), B41-b12 and B41-17b-CD4 (\u003cstrong\u003eh\u003c/strong\u003e). \u003cstrong\u003ei\u003c/strong\u003e Superposition of HR1C in AMC008-b12, CNE40-HmAb64, B41-b12. A, B, C, D denote closed, moderately open, partially open, and fully open states. CD4-bound states are denoted with ’.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6890430/v1/ee021d5f5ad4911672f17717.png"},{"id":86301959,"identity":"fd7c477b-d323-447d-bb6a-107892c1aa25","added_by":"auto","created_at":"2025-07-09 06:31:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1973401,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRemodeling of AMC008 SOSIP.v4.2 upon CD4 binding. a\u003c/strong\u003e 2.9 Å cryo-EM density map (left and middle, top and side views) and atomic model (right) of AMC008 in complex with CD4 D1D2. \u003cstrong\u003eb \u003c/strong\u003eAMC008-CD4 model fitted in a 3.6 Å unsharpened map. \u003cstrong\u003ec\u003c/strong\u003e Superimposition of gp41s of AMC008-b12 and AMC008-CD4. \u003cstrong\u003ed \u003c/strong\u003eSuperimposition of AMC008-CD4 with AMC008-VRC01-35O22, B41-17b-CD4, and AMC008-b12 shows remodeling of β20/β21 upon binding of CD4. Green dashed lines indicate contacts between the CD4 F43 residue and AMC008 gp120. \u003cstrong\u003ee\u003c/strong\u003e The W571\u003csub\u003egp41\u003c/sub\u003e residue becomes less buried upon binding of b12 or CD4. \u003cstrong\u003ef\u003c/strong\u003e Summary of structural features in gp41 conformational states with or without CD4 binding to its gp120 counterpart. CoRBS, coreceptor binding site; BS, bridging sheet.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6890430/v1/6aec7a76240c691d0157fa6f.png"},{"id":86301963,"identity":"578b6313-23d3-4631-912f-e08c384e24fb","added_by":"auto","created_at":"2025-07-09 06:31:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2938881,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRemodeling of AMC008 SOSIP.v4.2 upon b12 and 3BC315 binding. a\u003c/strong\u003e 3.7 Å cryo-EM density map (left and middle, top and side views) and atomic model (right) of AMC008 in complex with b12 and 3BC315 Fabs. A closed-up view shows interactions between 3BC315 light chain and b12 heavy chain.\u003cstrong\u003e b\u003c/strong\u003eComparison of the gp41s in AMC008-b12-3BC315 to the 3BC315-bound gp41 in AMC008-PGT121-VRC01-3BC315. Clashes are indicated by red * marks. 3BC315 binding epitope is circled. \u003cstrong\u003ec\u003c/strong\u003e Left: superimposition of AMC008-b12-3BC315, HT2-CD4 and BG505-E51-CD4 class II. Middle: Cα RMSD of HT2-CD4 in comparison with AMC008-b12-3BC315. Right: Cα RMSD of BG505-E51-CD4 class II in comparison with AMC008-b12-3BC315.\u003cstrong\u003e d\u003c/strong\u003e Superimposition of protomer 1 (left), 2 (middle), and 3 (right) of b12 and 3BC315-bound AMC008, two CD4-bound HT2, and three CD4-bound BG505. \u003cstrong\u003ee\u003c/strong\u003e Comparison of the HR1C conformations of protomer 1, 2 and 3 in \u003cstrong\u003eExtended Data Fig. h-l, o\u003c/strong\u003e to moderately open (AMC008-b12), symmetric partially open (6CM3), and symmetric fully open conformations (5VN3). \u003cstrong\u003ef \u003c/strong\u003eComparison of the distinct conformations between protomer C and protomer D. Close-up views show the different location and orientation of Trp571 side chain. \u003cstrong\u003eg \u003c/strong\u003eEstimation of transmembrane planes of CCR5s and CD4s in states C’ and D’. CD4-bound states are denoted with ’. Fabs and CD4s are hidden for better visualization in \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ed\u003c/strong\u003e and \u003cstrong\u003ef\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6890430/v1/ea80f43bbbfc82d89be87313.png"},{"id":86301961,"identity":"e63edb5f-f0c2-4ebb-8986-68391a51cc8c","added_by":"auto","created_at":"2025-07-09 06:31:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":573883,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding and neutralization cooperativity of b12 and 3BC315. a\u003c/strong\u003eMass shift of AMC008 SOSIP.v4.2 upon binding of excess b12, 3BC315, b12 \u0026amp; 3BC315, VRC01, and VRC01 \u0026amp; 3BC315. Increased molecular weights are normalized to number of Fab bindings by 55 kDa per Fab. Data is measured by mass photometry. P1 and P2, peak 1 and peak 2 of mass histogram. \u003cstrong\u003eb\u003c/strong\u003e-\u003cstrong\u003ec\u003c/strong\u003eNeutralization of HIV pseudoviruses BG505_T332N, SF162, BAL, TRO11 or MLV by varying concentrations of trimer-specific (\u003cstrong\u003eb\u003c/strong\u003e) b12 and (\u003cstrong\u003ec\u003c/strong\u003e) 3BC315. \u003cstrong\u003ed\u003c/strong\u003e-\u003cstrong\u003ef\u003c/strong\u003e Neutralization by b12 or 3BC315 alone at predetermined concentrations and the combination of b12 and 3BC315 against HIV pseudoviruses (\u003cstrong\u003ed\u003c/strong\u003e) BG505_T332N, (\u003cstrong\u003ee\u003c/strong\u003e)SF162, (\u003cstrong\u003ef\u003c/strong\u003e) BAL, (\u003cstrong\u003eg\u003c/strong\u003e) TRO11, and (\u003cstrong\u003eh\u003c/strong\u003e) MLV.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6890430/v1/69371c72fcba545070141078.png"},{"id":86301962,"identity":"d05fcd01-61ef-42e8-88d4-59b35711bd57","added_by":"auto","created_at":"2025-07-09 06:31:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1430660,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed mechanism of conformational changes from closed to fully open pre-CD4 bound and post-CD4 bound Env trimers. a\u003c/strong\u003e Mechanism of conformational changes from closed (AAA) to moderately open (BBB) to partially and asymmetric open (BCD, CCC, CCD, and CDD) and fully open (DDD). These conformations are capable of engaging CD4 and CD4-bound states are thus denoted with ’, e.g. AAA’, C’C’C’, etc. AAA\u003csub\u003eBR \u003c/sub\u003edenotes the closed Env states with one, two or three base-relaxed protomers. BCD\u003csub\u003eCD4\u003c/sub\u003e denotes one, two or three CD4-bound BBB or BCD states.\u003cstrong\u003e b\u003c/strong\u003e Representative models with the designated states for pre-CD4 bound conformations. \u003cstrong\u003ec\u003c/strong\u003e Representative models with the designated states for CD4-bound conformations. There are no existing models for CD4-bound BCD state.\u0026nbsp;\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6890430/v1/1a32fbca666dcdea2c0739a4.png"},{"id":109158091,"identity":"bbbf30c1-4828-453c-b87a-5eea4b7934dd","added_by":"auto","created_at":"2026-05-13 07:07:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13485383,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6890430/v1/0a7a5618-edea-4b66-976f-fa9b4fe493b9.pdf"},{"id":86302385,"identity":"823cc18c-7924-46a9-82a0-2576c6a9bf1e","added_by":"auto","created_at":"2025-07-09 06:39:41","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7612541,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"ExtendedDataFigureandtableandmovielegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-6890430/v1/18299f453416677a79ec23f7.docx"},{"id":86301958,"identity":"acdd9635-3bda-46a7-9750-6fba155796e8","added_by":"auto","created_at":"2025-07-09 06:31:41","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4157507,"visible":true,"origin":"","legend":"\u003cp\u003emovie1_V2_clash.mp4\u003c/p\u003e","description":"","filename":"movie1V2clash.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6890430/v1/8131bb0c51150a28002d12cb.mp4"},{"id":86301964,"identity":"86dc0b4e-2861-4afe-abe5-d390ed53ead1","added_by":"auto","created_at":"2025-07-09 06:31:41","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":5375499,"visible":true,"origin":"","legend":"\u003cp\u003emovie2_V2_noclash.mp4\u003c/p\u003e","description":"","filename":"movie2V2noclash.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6890430/v1/8d716c576e31c4804c114408.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Conformational Landscape of HIV-1 Env from Closed to Fully Open","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe HIV-1 envelope glycoprotein trimer (Env) is highly dynamic and essential for viral entry into host CD4\u003csup\u003e+\u003c/sup\u003e T cells\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Env is composed of three gp120 receptor binding subunits and three gp41 transmembrane subunits\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The fusion of viral and host membranes is initiated by receptor engagement of gp120 with human CD4, which induces conformational changes in Env that expose binding sites for coreceptors CCR5 or CXCR4\u003csup\u003e3,4\u003c/sup\u003e. Subsequent binding of these human coreceptors triggers insertion of the viral fusion peptide (FP) into the host membrane, eventually leading to viral genome entry, viral replication, and integration\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Due to the intrinsic instability of the HIV-1 Env, the SOSIP.664 modification was developed to stabilize soluble Env in its native-like state\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, enabling structural characterization of Env in both closed and CD4-bound open states\u003csup\u003e\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Comparative analyses of these states have revealed that CD4 binding triggers a cascade of structural rearrangements: burial of FP in a CD4-induced Env pocket, outward rotation of gp120, formation of a four-stranded bridging sheet and α0 helix, and displacement of V1V2 loops to expose the coreceptor binding site\u003csup\u003e\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. For clarity, we define the coreceptor binding site occluded closed conformational state as occluded closed, and the CD4-bound coreceptor binding site exposed conformational state as CD4-bound accessible open.\u003c/p\u003e\u003cp\u003eRecently, cryo-EM and cryo-ET studies have elucidated stepwise CD4 engagement with Env, providing biological relevance to the structural intermediates of SOSIP-stabilized Env\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. CD4 engagement begins with the binding of one CD4 molecule, resulting in an asymmetric open or closed conformational state of Env. Subsequent binding of a second or even a third CD4 molecule results in the asymmetric or symmetric opening of Env respectively, accompanied by a\u0026thinsp;~\u0026thinsp;50 \u0026Aring; reduction in viral-envelope-to-host-membrane distance. Notably, cryo-ET reveals that three CD4-bound Env adopts a partially open state distinct from the fully open state typically observed by cryo-EM\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Additionally, occluded open states \u0026ndash; exhibiting gp120 rotation but without V1V2 displacement, bridging sheet or α0 helix formation \u0026ndash; have been captured by antibody binding and characterized by both cryo-ET and cryo-EM\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. These occluded open states, alongside evidence that Env spontaneously samples open conformations without CD4\u003csup\u003e12,18\u0026ndash;21\u003c/sup\u003e, suggest that CD4 engages Env in a pre-existing conformational continuum.\u003c/p\u003e\u003cp\u003eTo delineate the interplay between occluded closed, occluded open, and accessible open states, we characterized novel conformations of AMC008 Env from a clade B Tier-1B virus. AMC008 Env has been reported as highly conformationally heterogeneous; however, SOSIP.v4.2 stabilization (I535M, L543N, A316W, H66R, and SOSIP.664) enables expression of soluble, flexible AMC008 SOSIP.v4.2 Env (hereafter AMC008)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. We identified novel Env conformations by complexing AMC008 with antibody 3BC315 (targeting the gp41-gp41 interface)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, antibody b12 (targeting the CD4 binding site; CD4bs)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, CD4, or a combination of 3BC315 and b12. 3BC315 and b12 were chosen based on prior insights: b12 facilitated the first cryo-EM structure of occluded open Env\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, while 3BC315 destabilizes Env via its interactions with the gp41-gp41 interface\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Cryo-EM structures of these complexes (resolutions ranging between 2.9\u0026ndash;3.9 \u0026Aring;), alongside an occluded closed reference structure determined to a resolution of 2.9 \u0026Aring;, unveiled distinct conformational states including a base-relaxed state upon 3BC315 binding, unique occluded moderately open states upon b12 or CD4 binding, and an asymmetric occluded open state upon combined b12 and 3BC315 binding. Integrating these results with previous studies, we propose a classification framework to characterize Env conformations and a mechanism by which HIV-1 Env transitions from occluded closed to accessible CD4-bound fully open en-route to membrane fusion. We observed enhanced 3BC315 binding occupancy in the presence of b12, and discovered that when engaging Env, antibodies 3BC315 and b12 interact with each other directly. Moreover, we decipher Env allosteric mechanisms underlying the cooperative accommodation of 3BC315 and b12, resulting in higher occupancy and increased neutralization. Our findings refine the mechanistic understanding of HIV-1 Env dynamics and provide a structural roadmap for targeting dynamic Env states in more potent designs of vaccines and immunotherapeutics.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003e3BC315 binds a quaternary epitope\u003c/b\u003e\u003c/p\u003e\u003cp\u003e3BC315 binding to Env was previously reported to dissociate Env trimers\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. To stabilize the 3BC315-AMC008 complex, we added VRC01 targeting the CD4bs and PGT121 targeting the V3-glycan epitope\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. We determined a cryo-EM structure of the resulting AMC008-PGT121-VRC01-3BC315 complex at 3.2 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In the data set, we observed two classes having complexed either one copy of 3BC315 or none at all, whereas both classes exhibited full VRC01 and PGT121 occupancy (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). This structure reveals atomic details of the 3BC315 epitope-paratope interface. To assess conformational changes in 3BC315 upon Env engagement, we superimposed the Env-bound 3BC315 Fab with its unliganded crystal structure (Cα RMSD\u0026thinsp;=\u0026thinsp;0.6 \u0026Aring;; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec)\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. While the two structures are highly similar, key differences include the side chain reorientation of F56\u003csub\u003eCDRH2\u003c/sub\u003e to accommodate the N88\u003csub\u003egp120\u003c/sub\u003e glycan and the side chain reorientation of R27\u003csub\u003eCDRL1\u003c/sub\u003e to hydrogen-bond with N543\u003csub\u003egp41\u003c/sub\u003e (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003e3BC315 binds predominantly to one gp41 subunit but exhibits additional interactions with gp120 (same protomer) and adjacent gp41. The epitope is bordered by N88\u003csub\u003egp120\u003c/sub\u003e and N625\u003csub\u003egp41\u003c/sub\u003e glycans (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The N88\u003csub\u003egp120\u003c/sub\u003e glycan is axially flipped 180\u0026deg; and tilted 70\u0026deg; toward the Env apex to accommodate 3BC315 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Both 3BC315 heavy and light chains interact extensively with AMC008 with buried surface areas of 915 \u0026Aring;\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and 503 \u0026Aring;\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e on the epitope, respectively (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). CDRH2, CDRH3, CDRL1 and CDRL3 of 3BC315 form polar interactions with the epitope to stabilize the binding of 3BC315. (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). A hydrophobic groove formed by six aromatic residues (CDRL1 Y30 and F32; CDRL3 Y91, Y94, Y100\u003csub\u003eE\u003c/sub\u003e, and F100\u003csub\u003eI\u003c/sub\u003e) accommodates an Env α-helix spanning the fusion peptide proximal region (FPPR) and the N-terminal region of heptad repeat 1 (HR1N) (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Additionally, the 3BC315 CDRH3 loop inserts into a cavity at the gp120-gp41-gp41 interface, positioning its tip at the hydrophobic gp120-gp41 interface (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003e\u003cb\u003e3BC315-bound base-relaxed AMC008\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe AMC008-PGT121-VRC01-3BC315 structure reveals remodeling of the Env base as compared to occluded closed Envs\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. To investigate this base remodeling, we solved a 2.9 \u0026Aring; cryo-EM structure of occluded closed AMC008 by complexing AMC008 with VRC01 Fab and 35O22 Fab (targeting the gp120/gp41 interface) (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003ea)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSuperimposition of the AMC008-PGT121-VRC01-3BC315 and occluded closed AMC008-VRC01-35O22 structures demonstrates dramatic displacements by more than 5 \u0026Aring; in epitope-associated structural elements upon 3BC315 binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). A hinge region comprised of residues L523\u003csub\u003egp41\u003c/sub\u003e-S534\u003csub\u003egp41\u003c/sub\u003e undergoes rigid-body movement, coupled with inward movement of FP toward the Env core and outward movement of HR1N (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb,c). Within the 3BC315-bound protomer, FP is embedded in a cavity formed by the non-helical α0 loop, the β0 strand, the α0-β0 connecting loop, and remodeled HR1N (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Furthermore, we aligned our 3BC315-bound gp41 to other gp41 remodeled Envs in the literature\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan additionalcitationids=\"CR30 CR31\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The 3BC315-bound gp41 resembles gp41s of T/F100-8ANC195 or CAP256.wk34.c80 SOSIP.RnS2, whereas, similar to our AMC008-PGT121-VRC01-3BC315, the FP burial occurs despite retention of occluded closed gp120 conformation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee,f). Notably, FP burial is a hallmark of Env opening\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In occluded partially open CNE40-HmAb64 and accessible partially open BG505-8ANC195-17b-CD4, FPs adopt conformations similar to 3BC315-bound gp41 but with FPs less extended to avoid clashes with the C-terminal region of HR1 (HR1C). In contrast, in occluded fully open B41-b12 and CD4-bound accessible fully open B41-17b-CD4, FP burial persists but with further retraction to accommodate HR1C (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Based on this data we conclude that FP conformation in 3BC315-bound AMC008 gp41 is equivalent to gp41 conformations in T/F100-8ANC195 and CAP256.wk34.c80 SOSIP.RnS2, and this conformation most resembles partially open states. Thus, we define this FP buried closed conformational state as the base-relaxed state. In contrast, FP of BG505-PGT122-VRC34.01 sits in an opposite orientation away from the Env core directed by antibody VRC34.01 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Taken together, these results underscore FP conformational plasticity across closed, partially open, and fully open Env states.\u003c/p\u003e\u003cp\u003eTo exclude potential confounding effects of VRC01/PGT121 on our observed base-relaxation, we determined cryo-EM structures of AMC008 bound solely to 3BC315. Unlike the AMC008-PGT121-VRC01-3BC315 complex that exhibited no more than single 3BC315 occupancy, the 3BC315-only dataset resolved two 3D classes: AMC008 bound to one (3.6 \u0026Aring;) or two (3.5 \u0026Aring;) 3BC315 Fabs (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb,\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003ed,\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). More importantly, all 3BC315-bound protomers adopt the base-relaxed conformation, while unbound protomers retain the closed conformation (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee), confirming that remodeling is driven by 3BC315 engagement rather than auxiliary antibodies.\u003c/p\u003e\u003cp\u003e\u003cb\u003eb12-bound moderately open AMC008\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe next determined the cryo-EM structure of AMC008 in complex with b12 at a resolution of 3.9 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Surprisingly, the degree of gp120 opening observed in the AMC008-b12 structure is significantly reduced compared to the previously resolved B41-b12 structure\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. To quantify gp120 openness in AMC008-b12, we measured interprotomer distances for representative Cα residues at the V3 base (H330\u003csub\u003egp120\u003c/sub\u003e), V1V2 base (P124\u003csub\u003egp120\u003c/sub\u003e), and CD4bs (D368\u003csub\u003egp120\u003c/sub\u003e). These measurements were compared across closed (AMC008-VRC01-35O22), partially open (CNE40-HmAb64, BG505-8ANC195-17b-CD4), and fully open (B41-b12, B41-17b-CD4) Env structures (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b,d-g)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. The AMC008-b12 openness is intermediate between closed and partially open states, leading us to define this as a moderately open state. Subsequent alignment of gp41 across these structures reveals distinct HR1C conformations: in partially and fully open states, the HR1C helix extends by at least one additional N-terminal turn and rotates in divergent directions relative to the closed state. While AMC008-b12 also exhibits N-terminal helical extension of HR1C, its orientation remains similar to a closed state (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Integrating gp120 openness, gp41 conformation, and CD4-binding status, we categorized Env states as follows: A (closed), A\u003csub\u003eBR\u003c/sub\u003e (base-relaxed closed), B (moderately open), C (partially open), C\u0026rsquo; (CD4-bound partially open), D (open) and D\u0026rsquo; (CD4-bound open).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNotably, significant displacement was observed at the β\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\stackrel{-}{4}\\)\u003c/span\u003e\u003c/span\u003e/β26 strands of the gp120 N- and C-termini, which insert into the gp41 \u0026lsquo;four-helix collar\u0026rsquo; \u0026ndash; identifying this region as a pivotal point for gp120 rotation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Displacements in the V1, V2, and V4 loops likely reflect inherent loop flexibility and isolate-specific sequence differences. Docking b12 Fab onto closed AMC008 reveals steric clashes with N197 and N301 glycans (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Upon b12 binding, the N197 glycan reorients to avoid clashes, accompanied by limited gp120 opening. This limited opening is sufficient for b12 accommodation, suggesting that SOSIP.v4.2-stabilized AMC008 samples open states less readily than B41 SOSIP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e\u003cp\u003eThe gp41 conformation in AMC008-b12 (state B) resembles gp41 of the closed state A, with solvent-exposed FP and bent FPPR/HR1N helix. However, HR1C in AMC008-b12 exhibits a 4.4\u0026deg; axial rotation toward the central symmetry axis and HR1N in AMC008-b12 is better resolved compared to state A (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Trimeric gp41 assemblies align closely between CD4-bound and unbound states for both partially open (C/C\u0026rsquo;) and fully open (D/D\u0026rsquo;) conformations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg,h). Notably, while HR1C conformations in states B, C, and D appear distinct when aligned by protomer, their overall helical bundle compactness is similar when aligning full trimeric assemblies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei).\u003c/p\u003e\u003cp\u003e\u003cb\u003eCD4-bound moderately open AMC008\u003c/b\u003e\u003c/p\u003e\u003cp\u003eObserving distinct conformational differences between occluded open AMC008-b12 and B41-b12 complexes, we hypothesized that CD4 binding to AMC008 would induce an alternative conformation. We therefore determined the cryo-EM structure of the AMC008-CD4 complex at a resolution of 2.9 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). Interprotomer distance measurements revealed that CD4-bound AMC008 exhibits intermediate gp120 openness, positioned between closed and partially open Envs but less open than AMC008-b12 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-g). CD4 binding destabilized interprotomer contacts at the trimer apex, rendering the V1V2V3 region more flexible and disordered (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Local classification of the apex (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003ee) yielded a 3.6 \u0026Aring; map showing that AMC008-CD4 gp120 retains occluded Env-like features: disordered but non-displaced V1V2 loops maintain the coreceptor binding site occluded despite partly disordering of V3, with no formation of the α0 helix or 4-stranded bridging sheet (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). This structural arrangement explains the finding that CD4 binding to AMC008 SOSIP.v4.2 only marginally enhances binding of 17b, an antibody targeting the coreceptor binding site\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Additionally, two previous reports identified single CD4-bound Envs in closed conformation with occluded coreceptor binding sites (state A\u0026rsquo;)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. As AMC008-CD4 retains gp41 assembly similarity to AMC008-b12 despite differing gp120 openness, we classify it as state B\u0026rsquo; (CD4-bound occluded moderately open) under our established criteria (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003eThe binding of CD4 does not result in the formation of a 4-stranded bridging sheet in AMC008-CD4, however, the coordinates of β20/β21 align well with other CD4-bound Env gp120s to form the essential contacts with the CD4 F43 residue, indicating the remodeling of β20/β21 is presumably the first step of CD4 engagement (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). This aligns with prior studies showing that restricting β20/β21 mobility via an interdomain 113C-429C disulfide bond impairs CD4 binding, underscoring the importance of β20/β21 remodeling for CD4 engagement\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Comparisons of the closed, the CD4-bound, and the b12-bound AMC008 structures reveal that W571\u003csub\u003egp41\u003c/sub\u003e transitions from deeply buried between α0\u0026ndash;1 and β\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\stackrel{-}{4}\\)\u003c/span\u003e\u003c/span\u003e (state A/A\u0026rsquo;) to exiting this hydrophobic pocket in states B/B\u0026rsquo; as a consequence of HR1C helix bundle compaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003eBased on this analysis, we find the HIV-1 Env trimer adopts distinct conformational states characterized by progressive gp120 opening and gp41 rearrangements. The occluded closed state A features tightly packed gp120 subunits at the trimer apex, solvent-exposed FP, bent FPPR/HR1N helices, and a non-extended HR1C bundle. A variant of this state, base-relaxed closed state A\u003csub\u003eBR\u003c/sub\u003e, retains most features of state A but adopts a gp41 base conformation resembling the partially open state C. In the occluded moderately open state B, gp120s rotate outward moderately, while gp41 retains state A-like features except for an extended and compacted HR1C bundle. Further opening defines the occluded partially open state C, where gp120s rotate more prominently, FP buries as a helical segment, and FPPR/HR1N adopts bent or straight conformations. The occluded fully open state D exhibits maximal gp120 rotation, a compact HR1C bundle, and FP repositioned into a shorter loop to avoid clashes. CD4 binding induces state-specific changes: it leaves state A\u0026rsquo; unaltered relative to state A, destabilizes the V1V2V3 apex in state B\u0026rsquo;, displaces V1V2 while forming the α0 helix and the 4-stranded bridging sheet to expose the coreceptor binding site in states C\u0026rsquo;/D\u0026rsquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003ef).\u003c/p\u003e\u003cp\u003e\u003cb\u003e3BC315 and b12-bound asymmetric open AMC008\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHaving observed alternative conformational changes upon binding either b12 or 3BC315, we rationalized that complexing AMC008 with both antibodies might lead to further structural rearrangements. We determined a cryo-EM structure of the AMC008-b12-3BC315 complex at a resolution of 3.7 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Unexpectedly, with the presence of b12, the binding occupancy of 3BC315 increased from one or two in our base-relaxed structures to three. Additionally, unlike the limited degree of gp120 opening observed in the AMC008-b12 complex, gp120s of the AMC008-b12-3BC315 complex are more open but in an asymmetric manner. Heavy chain framework region (C\u003csub\u003eH1\u003c/sub\u003e domain) of one b12 Fab interacts with light chain framework region (C\u003csub\u003eL\u003c/sub\u003e domain) of one 3BC315 Fab indicating a mechanism to enhance binding of the two antibodies by cooperativity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ea)\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Despite the global conformational opening of AMC008 and increased 3BC315 binding stoichiometry compared to our solely 3BC315 bound structures, there are only subtle local conformational changes in FP N-terminus and FPPR of the 3BC315 epitope. However, HR1C in each protomer rotates, becomes more compact, and forms additional helical turns, forcing FP to adopt a less extended conformation to prevent clashes with the rotated HR1C compared to the base-relaxed A\u003csub\u003eBR\u003c/sub\u003e state (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe next compared AMC008-b12-3BC315 to other published asymmetric open Env structures (Extended data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eh-p)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. This gp120 conformation of AMC008-3BC315-b12 is similar to two published CD4-bound asymmetric open Envs, HT2-CD4 and BG505-E51-CD4 class II, evaluated by measuring the interprotomer residue distances of gp120 in these structures and global alignments (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and Extended data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eh-j). Alignment of AMC008-b12-3BC315 to HT2-CD4 and BG505-E51-CD4 class II by protomer reveals that they are similar at each position regardless of CD4 binding. Despite these similarities, gp120s of AMC008-b12-3BC315 lack the features of a CD4-bound accessible open conformation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). We further compared gp41 conformations of AMC008-b12-3BC315 by aligning each protomer to the symmetric open reference structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). We observed that gp41 of each protomer can be matched with states C, or D, specifically, protomer 1 aligns well with state C while protomers 2 and 3 align better with state D. Therefore, we denote the b12 and 3BC315 bound AMC008 as state CDD. Furthermore, protomers of the currently existing asymmetric open Envs can be categorized using the same approach; thus, our state classification framework for symmetric Envs can be branched out to properly denote asymmetric Envs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ee and Extended Data Table\u0026nbsp;1).\u003c/p\u003e\u003cp\u003eFrom these models of asymmetric or symmetric Envs, we further differentiated protomeric states C and D based on the coordination of W571\u003csub\u003egp41\u003c/sub\u003e: the side chain of W571\u003csub\u003egp41\u003c/sub\u003e is above the α0-β0 connecting loop in state C; but is beneath the loop and is buried in a hydrophobic pocket formed by F53\u003csub\u003egp120\u003c/sub\u003e, P79\u003csub\u003egp120\u003c/sub\u003e, C218\u003csub\u003egp120\u003c/sub\u003e, and C247\u003csub\u003egp120\u003c/sub\u003e in state D (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Together with states A/B/B\u0026rsquo;, we identify W571\u003csub\u003egp41\u003c/sub\u003e to be a crucial residue that changes position corresponding to the conformational state of HIV-1 Env, consistent with previous structural and functional studies of Env W571\u003csub\u003egp41\u003c/sub\u003e\u003csup\u003e12,39,40\u003c/sup\u003e. To study the biological relevance of the states C\u0026rsquo; and D\u0026rsquo;, we docked Env models of states C\u0026rsquo;/D\u0026rsquo; into a cryo-ET density map of three CD4-bound Env, and then aligned gp120-CD4-CCR5 and AlphaFold-predicted full-length CD4 models to the docked Envs (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eg)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Using three copies of full-length CD4 and gp120-CD4-CCR5, we approximated the membrane surfaces of CCR5 and CD4. The approximated membrane surface of CD4 in state C\u0026rsquo; matches the membrane density, while state D\u0026rsquo; does not, consistent with the observation that Env in complex with CD4 imaged by cryo-ET adopts a partially open state\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. We discovered that the membrane surfaces for CCR5 and CD4 do not positionally match in state C\u0026rsquo; but appear to be much closer in D\u0026rsquo;. Based on this analysis, we predict that the virus-to-host membrane distance for state D\u0026rsquo; will likely be ~\u0026thinsp;110 \u0026Aring;, while the distance is measured to be ~\u0026thinsp;140 \u0026Aring; in state C\u0026rsquo; by cryo-ET\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Therefore, we expect states A\u0026rsquo;, B\u0026rsquo;, and C\u0026rsquo; all to be early states which do not engage the co-receptor, while state D\u0026rsquo; is likely primed to engage with the coreceptor and followed by membrane insertion of FP and dissociation of gp120.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStructural and functional cooperativity of b12 and 3BC315\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCryo-EM analysis of the AMC008-3BC315 complex revealed two distinct populations: 48.2% of particles were bound to a single 3BC315 Fab, while 51.8% accommodated two Fabs per trimer (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Strikingly, in the AMC008-PGT121-VRC01-3BC315 complex, 67.4% of particles retained one 3BC315 Fab, with the remaining 32.6% lacked 3BC315 entirely (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). In contrast, the AMC008-b12-3BC315 complex exhibited uniform occupancy, with three 3BC315 Fabs bound per trimer. These findings suggest that VRC01 and PGT121 stabilize Env in the occluded closed state (State A), which allosterically impedes 3BC315 binding, whereas b12 promotes a more open state (State B), enabling full 3BC315 occupancy. Moreover, as described above, simultaneous binding of b12 and 3BC315 facilitates stabilization of the complex through multiple hydrogen bonds formed between their constant framework regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eTo validate the cooperative binding of b12 and 3BC315 to Env, we performed mass photometry experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The molecular mass of trimeric native-like AMC008 was estimated at approximately 338 kDa. Upon binding excess 3BC315 Fab, the mass increased by 114 kDa (consistent with ~\u0026thinsp;2 Fabs), whereas excess b12 Fab or VRC01 Fab increased the mass by 170 kDa or 164 kDa, respectively (~\u0026thinsp;3 Fabs each). Consistent with our cryo-EM results, adding excess 3BC315 Fab to the AMC008-b12 complex induced an additional 162 kDa increase (~\u0026thinsp;3 Fabs). A distinct peak corresponding to AMC008-b12 without 3BC315 binding was observed, suggesting an all-or-none binding stoichiometry for 3BC315 Fab in the presence of b12. This implies that, in the context of b12, initial 3BC315 Fab binding markedly enhances affinity for the remaining two sites, indicative of positive cooperativity. In contrast, adding excess 3BC315 Fab to the AMC008-VRC01 complex yielded only a 66 kDa increase (~\u0026thinsp;1 Fab), reflecting negative cooperativity. These results align with our cryo-EM data, confirming that b12 facilitates 3BC315 binding to the AMC008 trimer, while VRC01 antagonizes it.\u003c/p\u003e\u003cp\u003eb12 is a CD4bs bNAb that exhibits limited binding and neutralization to tier 2 or higher HIV isolates, likely due to restricted conformational flexibility in these Env trimers. Our structures of AMC008-b12 and AMC008-b12-3BC315 reveal that while b12 binds to moderately open Env, the combination of both b12 and 3BC315 leads to an even more open Env conformation. We hypothesized that this cooperative binding, observed structurally, would enhance neutralization compared to either antibody alone, even against resistant isolates. To test this, we performed pseudovirus neutralization assays. 3BC315 neutralized all tested strains, whereas b12 only showed activity against SF162 and BaL, as previously reported and confirmed by our data (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eb,c)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. When the two antibodies were used in combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003ed-h), our neutralization assays showed a synergistic increase in neutralization activity, suggesting enhanced engagement of Env on the viral envelope. This neutralization synergy was pronounced for strains that could be neutralized by either b12 or 3BC315 \u0026ndash; SF162 and BaL (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee,f). Interestingly, even for strains completely resistant to b12 neutralization \u0026ndash; BG505_T332N and TRO11, the presence of b12 still enhanced the neutralization activity when in combination with 3BC315, though less significantly than against strains that can be neutralized by b12 or 3BC315 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003ed,g). These findings demonstrate that structural cooperativity between b12 and 3BC315 correlates with functional enhancement, with implications for antibody combination therapies.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we solved cryo-EM structures of base-relaxed asymmetric AMC008 with 3BC315 at atomic resolution, revealing conformational changes that resemble open Env conformations at the gp41 binding interface, namely FP, FPPR, and HR1N. Additionally, we solved structures of AMC008 bound with CD4 or b12 alone and with 3BC315. In our b12-bound or CD4-bound AMC008 structures, we found a new Env conformational intermediate, coined moderately open conformation, between the closed and partially open Env conformations with a moderately rotated gp120 and an N-terminally extended and more compacted HR1C. On the other hand, 3BC315 was found to induce a local gp41 conformational change without changing the conformation of gp120. Our structure of b12 and 3BC315 in complex with AMC008 revealed an alternative asymmetric occluded open Env conformation that resembles the two CD4-bound and three CD4-bound asymmetric open Env structures\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. We observe full occupancy (three copies of b12 and three copies of 3BC315 bound) in this asymmetric occluded open state, and we observe hydrogen-bonding ability between b12 C\u003csub\u003eH1\u003c/sub\u003e and 3BC315 C\u003csub\u003eL\u003c/sub\u003e constant IgG domains. These structural arrangements suggest a cooperativity mechanism between the two antibodies that combines framework antibody stacking ability as well as antibody manipulation of HIV-1 Env allostery to enhance combined antibody affinities to their respective epitopes. We confirmed our structural observations of antibody cooperativity using mass photometry and pseudovirus neutralization assays. Utilizing the effect of cooperativity between a combination of antibodies could be a potential advantage when developing prophylactic or therapeutic antibody cocktails leading to overall increased potency and breadth. Furthermore, 3BC315 exhibits the unique property of capturing an alternative gp41 conformation in a closed Env (AAA\u003csub\u003eBR\u003c/sub\u003e) that potentially shifts the conformational equilibrium of HIV-1 Env towards more open and immunogenic states. Developing vaccination strategies to elicit these types of broadly neutralizing antibodies could be another viable prophylactic avenue due to their ability to recover the increased neutralization susceptibility of Env toward neutralizing antibodies that bind open conformations.\u003c/p\u003e\u003cp\u003eUntil now, the conformational flexibility of Env has been challenging to comprehend, partially due to a lack of a systematic approach towards classifying intermediates from closed to open conformations, especially for pre-CD4 engaged conformations. Recent cryo-EM and cryo-ET studies have shown that asymmetric Env conformations are biologically relevant and exist in the context of one, two, or three CD4-bound Envs\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In addition, multiple smFRET studies have shown that unliganded Env on a native virion also spontaneously sample similar conformations that are enriched after CD4 engagement\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Furthermore, spontaneous opening of HIV-1 Env was observed in solution DEER spectroscopy, HDX-MS, and SAXS\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Therefore, we propose a model of the conformational trajectory of HIV-1 Env (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e6\u003c/span\u003e) that encompasses the trimeric conformational states that include closed (A), base-relaxed closed (A\u003csub\u003eBR\u003c/sub\u003e), moderately open (B), partially open (C), asymmetrically open (BCD, CCD, and CDD) and finally, fully open (D) states by categorizing each protomer of Env. In our proposed model, Env exhibits a natural ability to sample through stepwise opening from states A, B, C, and finally, D. Note that this conformational equilibrium is heavily skewed towards A for tier 2 and 3 isolates, while tier 1 HIV-1 isolates, such as B41, seem to sample these conformations equally as shown recently shown by DEER spectroscopy\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Although assigned as a closed state, local base-relaxation of FP/FPPR/HR1N may occur rarely and be captured by antibody binding\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Our AMC008-b12 complex is the first symmetric state B Env, and it is a crucial intermediate to delineate the transition from state A to state C. For Env to transition from closed to open conformations, it must assume state B to avoid the steric clash occurring at the C and D strands of the V2 loop (Movies 1 and 2). In line with our observations, SAXS studies suggest the existence of an early intermediate conformation of HIV-1 Env that involves increased flexibility around the apex loop contacts\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and smFRET studies further show that binding of one CD4 leads to the adjacent Env protomer sampling an intermediate conformational state between the closed state and CD4-bound open state\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. This conformational state in the protomer adjacent to the CD4-bound protomer most likely corresponds to state B of the present work. Our models of AMC008-b12 (B) and AMC008-CD4 (B\u0026rsquo;) provide the first structural evidence for this intermediate conformation. CD4 can engage Env at each state; however, the coreceptor binding site is only accessible in CD4-bound states C\u0026rsquo; and D\u0026rsquo;, but not in CD4-bound A\u0026rsquo; and B\u0026rsquo;. By CD4 and CCR5 membrane surface measurements, we tentatively conclude that the coreceptor engages Env in state D\u0026rsquo;; and states A\u0026rsquo;, B\u0026rsquo;, and C\u0026rsquo; are early intermediates of CD4 engagement (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). Thus, we hypothesize at least one protomer of Env in state D\u0026rsquo; is required for Env transition to postfusion. Our present model for the possible conformational states provides a framework of the conformational plasticity of HIV-1 Env and provides a template for therapeutic development and immunogen design strategies.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eDNA constructs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConstructs for transient expression of antibodies 35O22, VRC01, b12, and PGT121 in mammalian cells were obtained from the Kulp group at the Wistar Institute (Philadelphia, PA). Construct for expression of antibody 3BC315 in mammalian cells was designed in-house and synthesized by a commercial vendor (Genscript). The full-length BG505 plasmid was used to make point mutations at T332N for pseudotype virus production. Plasmids expressing HIV backbone ΔEnv (pSG3), SF162, BAL, TR011, and murine leukemia virus (MLV) control envelope were obtained from the NIH AIDS Reagents Program.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAMC008 SOSIP.v4.2 expression and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAMC008 SOSIP.v4.2 Env and furin were transiently co-transfected in Expi293F cells (Gibco) at a cell density of 2.5x10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003ecells/mL. The supernatant of the cell culture was harvested five days after transfection, or once cell viability was below 50%, by centrifugation at 6000g for 30 minutes, followed by 0.2 μm filtration and the addition of 1 mM PMSF. 0.5 mL lectin resin (Vector Laboratories) was added to the supernatant and incubated overnight. The overnight incubated supernatant was loaded and flowed through a gravity column (Bio-Rad), washed with 10 column volumes (CV) of PBS, and eluted with 5 CV of elution buffer (1M Methyl α-Mannopyranoside in PBS). The eluent was desalted by a PD-10 desalting column (Bio-Rad) and concentrated to 1 mL by centrifugal filter with a 100 kDa molecular weight cutoff (Millipore). The lectin-purified and desalted AMC008 Env protein was a mixture of AMC008 aggregate, trimer, and monomer. The mixture was purified by SEC using a 10/300 Superose 6 Increase column (Cytiva). Fractions corresponding to the AMC008 Env trimer were collected, concentrated using a centrifugal filter with a 100 kDa molecular weight cutoff to 1g/L, snap-frozen, and stored at -80 °C\u0026nbsp;for future usage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibody digestion and Fab purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe heavy chain and light chain of antibodies were transiently co-expressed in Expi293F cells with a ratio of 3:2. The supernatants of the cell cultures were harvested seven days after transfection or once cell viability was below 50% similarly to AMC008 Env. 1 mL Protein A resin (Cytiva) was added to the supernatant and incubated overnight. The overnight incubated supernatant was loaded onto a gravity column, washed with 10 CV of PBS, and eluted with 5 CV of elution buffer (20 mM sodium citrate, 150 mM sodium chloride, pH 2.5). Eluent was collected into a 50 mL centrifugal tube containing 5 mL of 1M pH 8 Tris to neutralize the elution buffer. Eluted antibodies were buffer-exchanged and concentrated to 5-10 mg/mL by centrifugal filtration with a 100 kDa molecular weight cutoff and stored at 4°C.\u003c/p\u003e\n\u003cp\u003eThe purified antibodies were diluted to around 1 g/L in digestion buffer (10 mM L-cysteine, 100 mM sodium acetate, 0.3 mM EDTA, pH 5.6). Papain (Millipore) was pre-incubated in digestion buffer at 37°C\u0026nbsp;for 10 minutes. The activated papain was added to the antibody and incubated at 37°C overnight. Digestions were terminated by adding 3 mM iodoacetamide. The digested mixtures containing Fab, Fc, and undigested antibody were purified by Protein A resin. 1 mL of Protein A resin was added to the mixtures and loaded onto a gravity column and incubated for 10 minutes. Flow-through was collected after incubation, concentrated, and buffer exchanged to PBS in a centrifugal filter with a 10 kDa molecular weight cutoff (Millipore). 10 CV of PBS was added to wash the column. 5 CV of Protein A elution buffer was added to elute Fc and undigested antibody. Fabs were stored at 4 °C for future usage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComplexation and cryo-EM sample preparation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComplexes were formed by incubating AMC008 Env with Fabs or CD4 in a 1:9 molar ratio in PBS for at least one hour at 4 °C. The mixture was purified by SEC using a 10/300 Superose 6 Increase column. The fractions corresponding to the complex were collected, combined, concentrated to 1 g/L, snap-frozen, and stored at -80 °C for future use.\u003c/p\u003e\n\u003cp\u003eComplexes were thawed, diluted to around 0.1 g/L, and placed on ice for cryo-grid preparation. GF-1.2/1.3-3Au-45nm cryo-EM grids (Protochips) were glow-discharged for 30 seconds, and the glow-discharged grids were coated with graphene oxide (GO). Complexes were vitrified by applying 4 μL diluted sample to the GO-coated grid using the vitrobot Mark IV (FEI) at 4 °C and 100% humidity, followed by plunging into liquid ethane.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCryo-electron microscopy\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003edata collection and processing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCryo-EM data were collected with a 300 kV Titan Krios G3i microscope equipped with a K3 Summit Direct Electron Detector camera at 81,000x magnification, a nominal dose of 58 e-/Å2, and a defocus range from -0.5\u0026nbsp;μm\u0026nbsp;to -2.5\u0026nbsp;μm. Movies were processed by RELION v3.1\u003csup\u003e45\u003c/sup\u003e using a standard cryo-EM data processing workflow. Workflow included motion correction, CTF estimation, LoG picking, particle extraction, 2D classification, manual selection of good 2D class averages, 3D classification and refinement, and Bayesian polishing. The polished particles were imported into CryoSPARC\u003csup\u003e46\u003c/sup\u003e for Non-uniform refinement. Consensus maps of AMC008-PGT121-VRC01-3BC315, AMC008-VRC01-35O22, AMC008-b12, AMC008-CD4, AMC008-3BC315, and AMC008-b12-3BC315 were generated with the workflow mentioned above (Extended Data Fig. 5, 6). In the AMC008-PGT121-VRC01-3BC315 dataset, we observed zero or one 3BC315 binding to the AMC008 SOSIP.v4.2 Env. We performed a classification focused on the 3BC315 binding site with three classes. Two classes that had 3BC315 binding were combined and reconstructed to the final AMC008-PGT121-VRC01-3BC315 map (Extended Data Fig. 5d).\u003c/p\u003e\n\u003cp\u003eIn the AMC008-CD4 dataset, we observed one to three CD4s binding to AMC008 SOSIP.v4.2. We simulated three density maps of one, two, and three CD4s binding to AMC008 SOSIP.v.4.2, and did a heterogeneous refinement with the simulated maps. The resulting 3D classes of AMC008-CD4 were all in the same conformational state despite the binding occupancy of CD4. We reconstructed the final map with particles of the three CD4-bound class and imposed C3 symmetry, as the three CD4-bound class was most populated and had the best view distribution. To investigate the V1V2V3 conformation of AMC008-CD4, we transferred particles of the three CD4-bound class back to Relion and performed a 3D classification focused on the Env apex with four classes. Two of the classes with density definition on Env apex were combined and imposed C3 symmetry to reconstruct a map with better density on Env apex.\u003c/p\u003e\n\u003cp\u003eIn the AMC008-VRC01-35O22 dataset, we observed exclusively three VRC01 Fabs binding to the Env, while one to three 35O22 Fabs bound to the AMC008 SOSIP.v4.2 Env when processing the cryo-EM data. Reconstructing each complex population with different 35O22 Fab binding occupancy, we found that the binding of 35O22 did not alter the AMC008 SOSIP.v4.2 Env conformation and only subtle differences were observed around the 35O22 epitope. After applying multiple data processing schemes, we found that combining populations with two or three 35O22 Fab binding occupancy and imposing C3 symmetry gave the best 3D reconstruction, considering both the global and local resolutions at the Env base (Extended Data Fig. 6a).\u003c/p\u003e\n\u003cp\u003eIn the AMC008-3BC315 dataset, we observed one or two 3BC315 binding to the AMC008 SOSIP.v4.2, and the ratio of the two populations was about 1:1. Reconstruction of the two classes showed dramatic conformational changes around the 3BC315 epitope. We performed a classification focused on the 3BC315 binding site with ten classes. We only included particles with high confidence in 3BC315 binding occupancy for the final round of reconstruction (Extended Data Fig. 6b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eModel building and refinement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCrystal structures of CD4 (PDB: 1WIO), VRC01 (PDB: 4LST), 35O22 (PDB: 4TOY), PGT121 (PDB: 4JY4), b12 (PDB: 1HZH), and 3BC315(PDB: 5CCK) were used as reference models. A reference model of AMC008 Env was generated by homology modeling using Modeler\u003csup\u003e47\u003c/sup\u003e. Reference models were rigid-body docked into the density maps in UCSF Chimera\u003csup\u003e48\u003c/sup\u003e and manually\u0026nbsp;rebuilt in Coot\u003csup\u003e49\u003c/sup\u003e. N-linked glycans were added to the models according to the N-linked glycosylation consensus sequence and the density maps. The manually rebuilt models were further refined using Rosetta FastRelax\u003csup\u003e50\u003c/sup\u003e. Refined models were reinspected and adjusted in Coot and refined by Rosetta again. These steps were repeated until no significant improvement was observed. Model geometry was validated by MolProbity\u003csup\u003e51\u003c/sup\u003e, glycan conformation was validated by Privateer\u003csup\u003e52\u003c/sup\u003e, and model fit-to-map was validated by EMRinger\u003csup\u003e53\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMass photometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eComplexes for mass photometry were prepared by mixing AMC008 Env with Fabs in a 1:9 molar ratio in PBS and incubating on ice for 30 minutes. Samples were diluted in PBS to optimal concentrations right before data collection. Data were collected by a TwoMP mass photometer (Refeyn) in default settings for 60 seconds and were calibrated by a ladder comprising bovine serum albumin (66 kDa), β-amylase (224 kDa), and thyroglobulin (670 kDa). Raw data were analyzed using DiscoverMP software to generate mass histograms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell lines and transfections for neutralization assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHEK 293 T cells (ATCC) and TZM-bl cells (NIH AIDS Reagents Program) were maintained in DMEM (ThermoFisher) supplemented with 10% heat-inactivated fetal bovine serum (Atlas Biologicals). Pseudotype viruses were produced as previously described\u003csup\u003e54\u003c/sup\u003e. Briefly, HEK 293T cells were co-transfected with 4 µg of a plasmid encoding the desired Env protein and 8 µg of a plasmid expressing the HIV-1 backbone Δ Env (pSG3ΔEnv − NIH AIDS Reagents) using GeneJammer (Aglient). Forty-eight hours after transfection, cell supernatants were harvested, filtered using a 45 µm filter, aliquoted, stored at −80°C and titered.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeutralization assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePseudotyped viruses were titered to yield 150,000 RLU after 48 h of infection with TZM-BL cells\u003csup\u003e54\u003c/sup\u003e. Monoclonal antibodies (MAbs) were serially diluted either individually or mixed together as combination at predetermined concentrations in 96-well plates followed by incubation with respective pseudotyped viruses before adding 10,000 TZM-BL cells (NIH AIDS Reagent Program) per well with dextran (ThermoFisher). Forty-eight hours post incubation, media was removed, and cells were lysed using BriteLite luciferase reagent (Revvity). Luminescence was then measured using the Synergy2 plate reader (BioTek Instruments).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistics and calculations were performed using GraphPad Prism 10.0. \u0026nbsp;MAb titer was determined for 50% virus neutralization (ID50), values were computed and graphed with a nonlinear regression model of percentage neutralization vs log10 concentration of MAbs.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe atomic models were deposited to PDB under accession codes 9NBT (AMC008-VRC01-35O22), 9NBY (AMC008-PGT121-VRC01-35022), 9NC0 (AMC008-b12), 9OAJ (AMC008-CD4), 9NC3 (AMC008-b12-3BC315), 9NC6 (AMC008-3BC315 (2x)) and 9NC8 (AMC008-3BC315 (1x)). The cryo-EM maps were deposited to EMDB under accession codes EMD-49236 (AMC008-VRC01-35O22), EMD-49238 (AMC008-PGT121-VRC01-35022), EMD-49239 (AMC008-b12), EMD-70287 (AMC008-CD4), EMD-49240 (AMC008-b12-3BC315), EMD-49241 (AMC008-3BC315 (2x)) and EMD-49242 (AMC008-3BC315 (1x)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Thomas Klose at the Purdue University Cryo-EM facility and Ruben Diaz Avalos at the La Jolla Institute for their assistance in sample screening and data collection. We thank Boyu Yin for his assistance in mass photometry experiments.\u003c/p\u003e\n\u003cp\u003eThis work was funded by W.W. Smith Charitable Foundation grant A2404 (to J.P.), NIH grant U19 AI166916 (to D.B.W and J.P). Funding sources were not involved in the design of this study, collection and analyses of data, decision to submit or preparation of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.C. and J.P. conceived experiments. J.C. produced protein samples. J.D. collected cryo-EM data. J.C., J.D., Z.L., and J.P. processed the cryo-EM data. J.C. built atomic models. J.C. and Z.L. performed mass photometry experiments. S.G. and R.S. performed pseudovirus neutralization assays. J.C. analyzed and interpreted the data. J.C., Z.L., and J.P. wrote the manuscript draft. S.G. contributed to the manuscript draft. All authors reviewed and commented on the manuscript. J.P. and D.B.W. supervised the work and acquired funding.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.B.W. notes several possible competing interests, which are managed by the Wistar General Council Office COI committee. These include consulting, BOD service, speaking, that can include in stock or monetary renumeration, and specific SRAs. Inovio Pharmaceuticals (BOD, consultant and SRA); AstraZeneca (speaker/consultant); Geneos (consultant \u0026amp; SRA); and possibly others that are managed by Wistar COI Committee. D.B.W. is a member of the International Society for Vaccines, AAI, ASGCT, AAAS, among other scientific societies. He also serves on NIH and NCI study sections and similar activities for other agencies.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWyatt, R. \u0026amp; Sodroski, J. The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e280\u003c/strong\u003e, 1884-8 (1998).\u003c/li\u003e\n\u003cli\u003eWard, A.B. \u0026amp; Wilson, I.A. The HIV-1 envelope glycoprotein structure: nailing down a moving target. \u003cem\u003eImmunol Rev\u003c/em\u003e \u003cstrong\u003e275\u003c/strong\u003e, 21-32 (2017).\u003c/li\u003e\n\u003cli\u003eKwong, P.D. et al. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e393\u003c/strong\u003e, 648-59 (1998).\u003c/li\u003e\n\u003cli\u003eBerger, E.A., Murphy, P.M. \u0026amp; Farber, J.M. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. \u003cem\u003eAnnu Rev Immunol\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 657-700 (1999).\u003c/li\u003e\n\u003cli\u003eBlumenthal, R., Durell, S. \u0026amp; Viard, M. HIV entry and envelope glycoprotein-mediated fusion. \u003cem\u003eJ Biol Chem\u003c/em\u003e \u003cstrong\u003e287\u003c/strong\u003e, 40841-9 (2012).\u003c/li\u003e\n\u003cli\u003eSanders, R.W. et al. A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies. \u003cem\u003ePLoS Pathog\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, e1003618 (2013).\u003c/li\u003e\n\u003cli\u003eJulien, J.P. et al. Crystal structure of a soluble cleaved HIV-1 envelope trimer. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e342\u003c/strong\u003e, 1477-83 (2013).\u003c/li\u003e\n\u003cli\u003eLyumkis, D. et al. Cryo-EM structure of a fully glycosylated soluble cleaved HIV-1 envelope trimer. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e342\u003c/strong\u003e, 1484-90 (2013).\u003c/li\u003e\n\u003cli\u003eKwon, Y.D. et al. Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env. \u003cem\u003eNat Struct Mol Biol\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 522-31 (2015).\u003c/li\u003e\n\u003cli\u003eOzorowski, G. et al. Open and closed structures reveal allostery and pliability in the HIV-1 envelope spike. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e547\u003c/strong\u003e, 360-363 (2017).\u003c/li\u003e\n\u003cli\u003eWang, H., Barnes, C.O., Yang, Z., Nussenzweig, M.C. \u0026amp; Bjorkman, P.J. Partially Open HIV-1 Envelope Structures Exhibit Conformational Changes Relevant for Coreceptor Binding and Fusion. \u003cem\u003eCell Host Microbe\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 579-592 e4 (2018).\u003c/li\u003e\n\u003cli\u003eYang, Z., Wang, H., Liu, A.Z., Gristick, H.B. \u0026amp; Bjorkman, P.J. Asymmetric opening of HIV-1 Env bound to CD4 and a coreceptor-mimicking antibody. \u003cem\u003eNat Struct Mol Biol\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 1167-1175 (2019).\u003c/li\u003e\n\u003cli\u003eDam, K.A., Fan, C., Yang, Z. \u0026amp; Bjorkman, P.J. Intermediate conformations of CD4-bound HIV-1 Env heterotrimers. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e623\u003c/strong\u003e, 1017-1025 (2023).\u003c/li\u003e\n\u003cli\u003eLi, W. et al. HIV-1 Env trimers asymmetrically engage CD4 receptors in membranes. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e623\u003c/strong\u003e, 1026-1033 (2023).\u003c/li\u003e\n\u003cli\u003eLiu, J., Bartesaghi, A., Borgnia, M.J., Sapiro, G. \u0026amp; Subramaniam, S. Molecular architecture of native HIV-1 gp120 trimers. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e455\u003c/strong\u003e, 109-13 (2008).\u003c/li\u003e\n\u003cli\u003eHarris, A.K., Bartesaghi, A., Milne, J.L. \u0026amp; Subramaniam, S. HIV-1 envelope glycoprotein trimers display open quaternary conformation when bound to the gp41 membrane-proximal external-region-directed broadly neutralizing antibody Z13e1. \u003cem\u003eJ Virol\u003c/em\u003e \u003cstrong\u003e87\u003c/strong\u003e, 7191-6 (2013).\u003c/li\u003e\n\u003cli\u003eYang, Z. et al. Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 732 (2022).\u003c/li\u003e\n\u003cli\u003eStadtmueller, B.M. et al. DEER Spectroscopy Measurements Reveal Multiple Conformations of HIV-1 SOSIP Envelopes that Show Similarities with Envelopes on Native Virions. \u003cem\u003eImmunity\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 235-246 e4 (2018).\u003c/li\u003e\n\u003cli\u003eLu, M. et al. Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e568\u003c/strong\u003e, 415-419 (2019).\u003c/li\u003e\n\u003cli\u003eBennett, A.L. et al. Microsecond dynamics control the HIV-1 Envelope conformation. \u003cem\u003eSci Adv\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, eadj0396 (2024).\u003c/li\u003e\n\u003cli\u003eHodge, E.A. et al. Structural dynamics reveal isolate-specific differences at neutralization epitopes on HIV Env. \u003cem\u003eiScience\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 104449 (2022).\u003c/li\u003e\n\u003cli\u003ede Taeye, S.W. et al. Immunogenicity of Stabilized HIV-1 Envelope Trimers with Reduced Exposure of Non-neutralizing Epitopes. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e163\u003c/strong\u003e, 1702-15 (2015).\u003c/li\u003e\n\u003cli\u003eKlein, F. et al. Broad neutralization by a combination of antibodies recognizing the CD4 binding site and a new conformational epitope on the HIV-1 envelope protein. \u003cem\u003eJ Exp Med\u003c/em\u003e \u003cstrong\u003e209\u003c/strong\u003e, 1469-79 (2012).\u003c/li\u003e\n\u003cli\u003eBurton, D.R. et al. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e266\u003c/strong\u003e, 1024-7 (1994).\u003c/li\u003e\n\u003cli\u003eLee, J.H. et al. Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 8167 (2015).\u003c/li\u003e\n\u003cli\u003eZhou, T. et al. Structural basis for broad and potent neutralization of HIV-1 by antibody VRC01. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e329\u003c/strong\u003e, 811-7 (2010).\u003c/li\u003e\n\u003cli\u003eJulien, J.P. et al. Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans. \u003cem\u003ePLoS Pathog\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, e1003342 (2013).\u003c/li\u003e\n\u003cli\u003eHuang, J. et al. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e515\u003c/strong\u003e, 138-42 (2014).\u003c/li\u003e\n\u003cli\u003eKong, R. et al. Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e352\u003c/strong\u003e, 828-33 (2016).\u003c/li\u003e\n\u003cli\u003eAnanthaswamy, N. et al. A sequestered fusion peptide in the structure of an HIV-1 transmitted founder envelope trimer. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 873 (2019).\u003c/li\u003e\n\u003cli\u003eGorman, J. et al. Structure of Super-Potent Antibody CAP256-VRC26.25 in Complex with HIV-1 Envelope Reveals a Combined Mode of Trimer-Apex Recognition. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e31\u003c/strong\u003e, 107488 (2020).\u003c/li\u003e\n\u003cli\u003eWang, S. et al. Human CD4-binding site antibody elicited by polyvalent DNA prime-protein boost vaccine neutralizes cross-clade tier-2-HIV strains. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 4301 (2024).\u003c/li\u003e\n\u003cli\u003eLiu, Q. et al. Quaternary contact in the initial interaction of CD4 with the HIV-1 envelope trimer. \u003cem\u003eNat Struct Mol Biol\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e, 370-378 (2017).\u003c/li\u003e\n\u003cli\u003eZhang, P. et al. Design of soluble HIV-1 envelope trimers free of covalent gp120-gp41 bonds with prevalent native-like conformation. \u003cem\u003eCell Rep\u003c/em\u003e \u003cstrong\u003e43\u003c/strong\u003e, 114518 (2024).\u003c/li\u003e\n\u003cli\u003eOyen, D. et al. Cryo-EM structure of P. falciparum circumsporozoite protein with a vaccine-elicited antibody is stabilized by somatically mutated inter-Fab contacts. \u003cem\u003eSci Adv\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, eaau8529 (2018).\u003c/li\u003e\n\u003cli\u003eParzych, E.M. et al. DNA-delivered antibody cocktail exhibits improved pharmacokinetics and confers prophylactic protection against SARS-CoV-2. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, 5886 (2022).\u003c/li\u003e\n\u003cli\u003eJette, C.A. et al. Cryo-EM structures of HIV-1 trimer bound to CD4-mimetics BNM-III-170 and M48U1 adopt a CD4-bound open conformation. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 1950 (2021).\u003c/li\u003e\n\u003cli\u003eWang, H. et al. Potent and broad HIV-1 neutralization in fusion peptide-primed SHIV-infected macaques. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e187\u003c/strong\u003e, 7214-7231 e23 (2024).\u003c/li\u003e\n\u003cli\u003eMo, H. et al. Conserved residues in the coiled-coil pocket of human immunodeficiency virus type 1 gp41 are essential for viral replication and interhelical interaction. \u003cem\u003eVirology\u003c/em\u003e \u003cstrong\u003e329\u003c/strong\u003e, 319-27 (2004).\u003c/li\u003e\n\u003cli\u003eHenderson, R. et al. Disruption of the HIV-1 Envelope allosteric network blocks CD4-induced rearrangements. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 520 (2020).\u003c/li\u003e\n\u003cli\u003eShaik, M.M. et al. Structural basis of coreceptor recognition by HIV-1 envelope spike. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e565\u003c/strong\u003e, 318-323 (2019).\u003c/li\u003e\n\u003cli\u003eJumper, J. et al. Highly accurate protein structure prediction with AlphaFold. \u003cem\u003eNature\u003c/em\u003e \u003cstrong\u003e596\u003c/strong\u003e, 583-589 (2021).\u003c/li\u003e\n\u003cli\u003eMunro, J.B. et al. Conformational dynamics of single HIV-1 envelope trimers on the surface of native virions. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e346\u003c/strong\u003e, 759-63 (2014).\u003c/li\u003e\n\u003cli\u003eMa, X. et al. HIV-1 Env trimer opens through an asymmetric intermediate in which individual protomers adopt distinct conformations. \u003cem\u003eElife\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e(2018).\u003c/li\u003e\n\u003cli\u003eZivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. \u003cem\u003eeLife\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, e42166 (2018).\u003c/li\u003e\n\u003cli\u003ePunjani, A., Rubinstein, J.L., Fleet, D.J. \u0026amp; Brubaker, M.A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. \u003cem\u003eNat Methods\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 290-296 (2017).\u003c/li\u003e\n\u003cli\u003e\u0026Scaron;ali, A. \u0026amp; Blundell, T.L. Comparative Protein Modelling by Satisfaction of Spatial Restraints. \u003cem\u003eJournal of Molecular Biology\u003c/em\u003e \u003cstrong\u003e234\u003c/strong\u003e, 779-815 (1993).\u003c/li\u003e\n\u003cli\u003eMeng, E.C. et al. UCSF ChimeraX: Tools for structure building and analysis. \u003cem\u003eProtein Science\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e, e4792 (2023).\u003c/li\u003e\n\u003cli\u003eEmsley, P. \u0026amp; Cowtan, K. Coot: Model-building tools for molecular graphics. \u003cem\u003eActa Crystallographica Section D: Biological Crystallography\u003c/em\u003e \u003cstrong\u003e60\u003c/strong\u003e, 2126-2132 (2004).\u003c/li\u003e\n\u003cli\u003eTyka, M.D. et al. Alternate states of proteins revealed by detailed energy landscape mapping. \u003cem\u003eJ Mol Biol\u003c/em\u003e \u003cstrong\u003e405\u003c/strong\u003e, 607-18 (2011).\u003c/li\u003e\n\u003cli\u003eChen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. \u003cem\u003eActa Crystallographica Section D\u003c/em\u003e \u003cstrong\u003e66\u003c/strong\u003e, 12-21 (2010).\u003c/li\u003e\n\u003cli\u003eAgirre, J. et al. Privateer: software for the conformational validation of carbohydrate structures. \u003cem\u003eNature Structural \u0026amp; Molecular Biology\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 833-834 (2015).\u003c/li\u003e\n\u003cli\u003eBarad, B.A. et al. EMRinger: side chain\u0026ndash;directed model and map validation for 3D cryo-electron microscopy. \u003cem\u003eNature Methods\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 943-946 (2015).\u003c/li\u003e\n\u003cli\u003eSarzotti-Kelsoe, M. et al. Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1. \u003cem\u003eJ Immunol Methods\u003c/em\u003e \u003cstrong\u003e409\u003c/strong\u003e, 131-46 (2014).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6890430/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6890430/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe molecular mechanism of HIV-1 entry into host cells is governed by dynamic conformational changes to its envelope glycoprotein (Env), which are triggered by the engagement of the host receptor CD4 and coreceptors. Structural insights into these transitions have been advanced by cryo-electron tomography (cryo-ET), resolving Env structures in closed and multifarious open states within native membranes, and by cryo-electron microscopy (cryo-EM), which has provided atomic details of these states. In this study, we determined cryo-EM structures of soluble native-like Env in complex with antibody 3BC315, antibody b12, CD4, or a combination of 3BC315 and b12. These combination studies allowed us to capture previously uncharacterized HIV Env conformational states. We observed enhanced 3BC315 binding occupancy in the presence of b12 and discovered that when engaging Env, antibodies 3BC315 and b12 interact with each other directly. Moreover, we decipher the allosteric mechanisms of Env, resulting in the cooperative accommodation of 3BC315 and b12, which leads to higher occupancy and increased neutralization potency. Integrating these novel states with the literature, we establish a classification framework for symmetric and asymmetric Env states, categorizing by their degree of openness and stepwise structural rearrangements. Our findings refine the mechanistic understanding of HIV-1 Env dynamics and provide a structural roadmap for targeting dynamic Env states for more potent designs of vaccines and immunotherapeutics.\u003c/p\u003e","manuscriptTitle":"Conformational Landscape of HIV-1 Env from Closed to Fully Open","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-09 06:31:36","doi":"10.21203/rs.3.rs-6890430/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ea94262d-dd0f-4789-8921-7aeeb3876a03","owner":[],"postedDate":"July 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":50878861,"name":"Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy"},{"id":50878862,"name":"Biological sciences/Immunology/Infectious diseases/HIV infections"},{"id":50878863,"name":"Biological sciences/Microbiology/Virology/Retrovirus"},{"id":50878864,"name":"Biological sciences/Biochemistry/Proteins/Glycoproteins"},{"id":50878865,"name":"Biological sciences/Microbiology/Vaccines/Protein vaccines"}],"tags":[],"updatedAt":"2026-05-13T07:07:15+00:00","versionOfRecord":{"articleIdentity":"rs-6890430","link":"https://doi.org/10.1038/s41467-026-69921-z","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-02-24 05:00:00","publishedOnDateReadable":"February 24th, 2026"},"versionCreatedAt":"2025-07-09 06:31:36","video":"","vorDoi":"10.1038/s41467-026-69921-z","vorDoiUrl":"https://doi.org/10.1038/s41467-026-69921-z","workflowStages":[]},"version":"v1","identity":"rs-6890430","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6890430","identity":"rs-6890430","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}