A DARPin Increases the Catalytic Activity of Botulinum Neurotoxin A1

A DARPin Increases the Catalytic Activity of Botulinum Neurotoxin A1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A DARPin Increases the Catalytic Activity of Botulinum Neurotoxin A1 Oneda Leka, Yufan Wu, Giulia Zanetti, Sven Furler, Thomas Reinberg, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2411408/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Dec, 2023 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract In this study, we characterized Designed Ankyrin Repat Proteins (DARPins) as investigative tools to probe botulinum neurotoxin (BoNT) function and as potential antidotes for botulism. We selected DARPins against the catalytic domain of BoNT/A1 and characterized them by biochemical, biophysical and structural studies in combination with functional assays in cultured neurons and muscle tissue. We identified DARPin-F5 that completely blocks SNAP25 substrate cleavage by BoNT/A1 in vitro . X-ray crystallography revealed that DARPin-F5 inhibits BoNT/A1 activity by interacting with a substrate-binding region between the α- and β-exosite. This DARPin blocked substrate cleavage of BoNT/A1 but not of BoNT/A3, indicating that DARPin-F5 is a subtype-specific inhibitor. We found that BoNT/A1 Glu-171 plays a critical role in the interaction with DARPin-F5 and its mutation to Asp, the residue found in BoNT/A3, resulted in a loss of inhibition of substrate cleavage by reducing DARPin affinity from pM to µM. In contrast to the in vitro results, DARPin-F5 increased BoNT/A1 activity in primary neurons. This result was confirmed by the mouse phrenic nerve hemidiaphragm assay, demonstrating faster paralysis in the presence of the DARPin. We show by functional studies in neuronal cells that DARPin-F5 increases translocation of the toxin. Our findings could have important implications for the identification of BoNTs in clinical samples as well as the development of excipients that allow BoNT treatment at a lower dosage and thereby prevent the generation of antibodies against the toxin. Biological sciences/Structural biology/X-ray crystallography Biological sciences/Biochemistry/Enzymes/Proteases Biological sciences/Cell biology/Protein transport Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Botulinum neurotoxins (BoNTs) produced by anaerobic bacteria of the genus Clostridium are the most poisonous bacterial protein toxins known [ 1 – 4 ]. BoNT intoxication in vertebrates causes botulism, a potentially life-threatening neuroparalytic syndrome. [ 5 ]. Therefore the toxins represent potential biological weapons. Despite their toxicity and as a result of their characteristics, including biological effectiveness and long persistence of action in patients, BoNTs are nowadays the most widely used therapeutic proteins in various human neurological and non-neurological disorders [ 6 , 7 ]. Furthermore, they are used in cosmetic applications. Traditionally, BoNTs are classified as seven serologically distinct proteins, referred to as BoNT/A through BoNT/G, however, their genetic variability is further increased by the existence of more than 40 subtypes within serotypes A, B, E and F [ 8 , 9 ]. Although many subtypes have not yet been well characterized, it was shown that some differ from the other proteins within a serotype with respect to their catalytic properties, substrate specificity, duration of action, and efficiency to enter neuronal cells [ 10 – 13 ]. Because they could outperform conventionally used BoNTs with respect to their biological activities, it appears important to characterize all BoNT subtypes. Furthermore, there exist mosaic toxins between serotypes C and D [ 14 ] and partially between A and F [ 15 ]. Recently, several BoNT-like sequences have been discovered in single bacterial strains whose occurrence is of great concern, because it indicates that Clostridium botulinum is able to horizontally transfer BoNT genes to other bacterial species [ 16 – 19 ]. BoNTs are produced as single precursors that share a common domain organization [ 20 – 23 ]. They are processed into mature toxins by cleavage into a ~ 50 kDa light chain (LC) and a ~ 100 kDa heavy chain (HC) that remain connected through an interchain disulfide bond. The LC is a zinc-dependent endopeptidase that specifically cleaves members of the soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) family of proteins, which are key components of the vesicular fusion machinery within presynaptic nerve terminals. BoNT/A and E cleave synaptosomal-associated protein 25 (SNAP25), and BoNT/B, D, F and G cut vesicle-associated membrane protein (VAMP). BoNT/C can cleave two substrates, SNAP25 and syntaxin [ 1 , 24 , 25 ]. SNARE cleavage blocks acetylcholine release at the neuromuscular junction, leading to a flaccid paralysis of muscles [ 5 ]. The HC consists of a 50 kDa N-terminal translocation domain (H N ) and a C-terminal receptor-binding domain (H C ) of similar size. A dual interaction mode involving two different receptors seems to be required for the neuro-specific binding of BoNTs [ 26 ]. Most BoNTs bind a polysialoganglioside (PSG) and synaptotagmin (Syt) or synaptic vesicle glycoprotein 2 (SV2) [ 1 , 3 , 27 ]. Upon binding to neuronal cell receptors, BoNTs are endocytosed into synaptic vesicles [ 1 ]. The low pH and the lipid environment within the synaptic vesicle is believed to initiate conformational changes in H N [ 4 , 28 ]. Amongst those, exposure of a hydrophobic peptide through a viral-fusion-peptide-like pH-dependent molecular switch seems to initiate the first step of membrane insertion of BoNT/A1 [ 29 ]. Subsequent conformational changes are thought to lead to the formation of an ion-conductive transmembrane channel through which the LC is translocated across the membrane. Once inside the reducing intracellular environment of the cytosol, the disulfide bond is reduced, resulting in release of the protease. The existence of multiple BoNT and BoNT-like toxin molecules represents a great public health threat, because they can potentially all cause botulism or be engaged as bioweapons. As result of this public health concern, multiple strategies have been developed for the treatment and prevention of botulism [ 30 , 31 ]. Like the toxoid-based tetanus vaccination against the structurally related Tetanus neurotoxin (TeNT), vaccination using BoNT toxoid efficiently protects against botulism [ 30 ]. Although different next-generation vaccines are being developed to replace the discontinued toxoid vaccine [ 32 ], vaccination is seldom used because botulism is a rare disease with less than 200 cases reported per year [ 30 ]. More importantly, vaccination would have a negative impact on the therapeutic benefits of BoNTs for medical applications. As a result, passive immunotherapy using polyclonal antibody preparations and sera, individual monoclonal antibodies and monoclonal antibody combinations are used for the medical treatment of botulism [ 30 ]. In addition, alternative strategies such as the application of camelid single-chain antibodies are also being developed [ 33 – 38 ]. A class of proteins whose usefulness as BoNT-inhibiting agents has not yet been explored are the DARPins (designed ankyrin repeat proteins) [ 39 ]. DARPins are small, engineered antibody mimetics derived from synthetic libraries of consensus ankyrin proteins that are very specific and show very high affinity to the target protein. They are successfully used in various research, diagnostic and therapeutic applications. Considerable benefits of DARPins are their small size of 14 or 18 kDa (4 or 5 ankyrin repeats of which two or three carry a randomized surface and are flanked by N-capping and C-capping repeats) and their high stability and solubility. In addition, DARPins can be generated rapidly and cost-efficiently in a soluble form and high yields in the cytoplasm of E. coli . Biomolecules like nanobodies and DARPins might contribute significantly to the development and of next-generation biomolecular antidotes against botulism. However, since they typically recognize conformational epitopes, nanobodies and DARPins also represent invaluable tools to probe the structure and function of BoNTs. In this study, we investigated the impact of DARPins selected against LC/A1 on toxin activity using biochemical and biophysical methods, X-ray crystallography and functional assays performed in vitro and in cells and tissue. Results Generation and characterization of DARPins selected against LC/A1 DARPins were selected over four rounds of ribosome display [ 40 – 42 ] using a semi-automated 96 well platform against full-length LC/A1 (amino-acid residues Pro-2 to Lys-448). In total for each selection 380 single clones were screened using a 384 well-based HTRF assay. From the initial hits, 32 were sequenced. For the selection against full-length LC/A1, 25 specific and unique clones were identified. These were expressed and IMAC purified. Binding was confirmed using ELISA. DARPin-F5 inhibits the catalytic activity of LC/A1 in vitro Because its C-terminus contributes to catalysis, the full-length LC represents the relevant target for BoNT/A1 inhibitor studies [ 35 , 43 , 44 ]. In a first step, we were searching for DARPins that inhibit the protease activity of full-length LC/A1. To this end, we incubated each of the 25 selected DARPins individually at different ratios with LC/A1 and monitored the cleavage of the recombinant human SNAP-25 substrate peptide spanning amino-acid residues Met-141 to Gly-204. We identified six DARPins that completely or partially inhibited the proteolytic activity of BoNT LC/A1. Among the candidates, DARPin 008-829-2386-F5 (named DARPin-F5 for short) was able to completely inhibit substrate cleavage at a DARPin/toxin molar ratio of 2.5:1 (Fig. 1 a) while the remaining five DARPins showed partial inhibition of LC/A1 enzymatic activity to varying degrees (data not shown). Because the C-terminus of the catalytic domain has been implicated in enzyme/substrate interactions [ 35 , 44 ], we also tested the ability of DARPin-F5 to prevent the cleavage of the SNAP25 peptide substrate using two C-terminally truncated LC/A1 variants (residues Pro-2 to Gly-421 and Pro-2 to Gly-433). DARPin-F5 also completely inhibited proteolytic activity of both truncated LC/A1 variants (not shown), indicating that DARPin-F5 doesn’t bind to the C-terminus of the catalytic domain. In full-length BoNT/A1 the catalytic domain is surrounded by the belt region, which is considered a pseudo-substrate that prevents LC/A1 protease activity before its delivery into the neuronal cytosol [ 45 ]. Although it is unknown if LC and HC remain bound to each other after reduction of the connecting disulfide bridge, reduced full-length BoNT/A1 efficiently cleaves SNAP25. As observed for LC/A1, DARPin-F5 was found to also completely inhibit reduced full-length BoNT/A1 (Fig. 1 b). Crystal structure of the LC/A1-DARPin-F5 complex To understand at the molecular level how DARPin-F5 inhibits the catalytic activity of LC/A1, we next focused on determining the crystal structure of the complex. Attempts to crystallize DARPin-F5 with the full-length catalytic domain or a slightly shorter LC/A1 variant (amino-acid residues Pro-2 to Gly-433) were not successful, probably because these variants are too flexible for crystallization and prone to aggregation [ 43 , 46 ]. Therefore, we focused on an even more truncated LC/A1 variant spanning amino-acid residues Pro-2 to Gly-421, which was previously used to solve the crystal structure of a LC/A1-SNAP25 toxin/substrate complex [ 47 ]. Crystals suitable for structural work were obtained within 1–2 weeks and the structure of the LC/A1-DARPin-F5 complex was solved at a resolution of 2.5 Å (Fig. 2 a and Supplementary Table S1; PDB code 8HKH). The crystal structure contains two toxin/DARPin complexes per asymmetric unit (rmsd value of 0.2 Å for 510 Cα atoms of both AC and BD complexes). In the following, we analyzed the AC complex. In botulinum neurotoxins, SNAP25 wraps around the catalytic domain in a manner very similar to the belt [ 47 ]. The binding interface includes the α-exosite and the β-exosite, two major structural motifs that bind the ~ 60 residue-long substrate remote from the catalytic site. It has been shown that the exosites play important roles for substrate binding and specificity [ 47 ]. The crystal structure of the complex reveals that DARPin-F5 binds to a substrate-binding site between the α- and β-exosites of LC/A1, far away from the active site of the catalytic domain. The DARPin therefore inhibits SNAP25 hydrolysis by preventing substrate binding. Consistent with this observation, it has been shown that nanobodies preventing the binding of the substrate also show potent inhibition of LC/A1 [ 35 , 36 ]. All BoNT-LCs share a globular domain fold in which a conserved substrate-binding groove extends from the catalytic site around the enzyme [ 47 ]. Loops 50, 170, 250, and 370 form the boundaries of this large cleft on the enzyme surface that in the neurotoxin holostructure is occupied by the belt. Superimposition of the LC/A1-DARPin-F5 complex structure with the structure of LC/A1 bound to SNAP25 (PDB code 1XTG) [ 47 ] shows that DARPin-F5 binds to the region of loop 170, thereby preventing the interaction of SNAP25 with this site (Fig. 2 b). The belt binds to LC/A1 in the full-length toxin in a manner that is very similar to the interaction of LC/A1 with the substrate [ 45 ]. Accordingly, DARPin could also interfere with the binding of the belt as shown in the superimposition of the LC/A1-DARPin-F5 complex structure with the structure of BoNT/A1 holotoxin (PBD 3BTA) (Fig. 2 c). The most prominent feature seen in the complex structure is a network of interactions involving Glu-171 of LC/A1 and three residues from different loops of the DARPin (Fig. 3 a). These interactions include a salt bridge that is formed between Glu-171 of LC/A1 and Lys-91 of the DARPin, respectively. In addition, Arg-25, and the phenolic hydroxy group of Tyr-50 of DARPin-F5 form hydrogen bonds with the backbone oxygen and the carbonyl group of Glu-171, respectively. Notably, Arg-25 is conserved amongst DARPins. Based on structural considerations, Glu-171 of LC/A1 seems to be a key residue for the interaction with DARPin-F5, although it is not directly involved in the interaction with the SNAP25 substrate. Moreover, two additional residues from LC/A1, Lys-128 and Asp-131, are also involved in the complex formation with DARPin-F5. The interactions seen at the interface are summarized in Supplementary Table S2. In addition, the 6xHis tag present in the DARPin (chains C and D) also interacts with the LC/A1 (chains B and A, respectively) of the other complex (Supplementary Figure S1). Residues His-11 to Gly-13 form a short β-strand that interacts in an antiparallel manner with the β-strand formed by residues Gly-255 to Phe-260. As a result of this interaction, the N-terminus (His-7 to His-10) points deep into the active site and extends towards the catalytic zinc. This second interaction is unlikely to contribute to the inhibition of LC/A1, because there is no evidence of tetrameric complexes in solution (Supplementary Figure S2). The interaction is therefore probably needed to establish the crystal packing. DARPin-F5 is a subtype-specific inhibitor of BoNT/A catalysis As shown in the alignment of Fig. 3 b, the DARPin-binding region is well conserved amongst BoNT/A subtypes. Glu-171 of LC/A1 is a conserved Asp in all other subtypes except BoNT/A6. The other two interacting residues, LC/A1 Lys-128 and Asp-131, are identical in all subtypes. The conservation of the DARPin-binding region suggests that DARPin-F5 might inhibit the catalytic activity of all eight BoNT/A subtypes. To test this hypothesis, we assesed whether DARPin-F5 is able to block the enzymatic activity of LC/A3, a potentially very attractive subtype for medical applications such as pain relief and orthopedics where a shorter onset and duration of action are required [ 13 , 48 ]. We found that DARPin-F5 was not able to inhibit SNAP25 catalysis by LC/A3. Therefore, we next focused on the role of Glu-171 in the inhibition of SNAP25 catalysis and generated LC/A1 and LC/A3 mutants, in which Glu-171 and Asp-171 were exchanged between subtypes. First, we tested the activity of the mutants: LC/A1-Glu171Asp and LC/A3-Asp171Glu cleaved SNAP25 with the same efficiency as the respective wild-type proteins. DARPin-F5 completely blocked SNAP25 cleavage by LC/A3-Asp171Glu (DARPin-F5/toxin molar ratio of 5:1 or 10:1) but did not inhibit substrate catalysis of LC/A1 Glu171Asp (Fig. 3 c), demonstrating the importance of this amino-acid residue for DARPin-F5-mediated inhibition of proteolysis. Taken together, our findings demonstrate that we can generate BoNT/A subtype-specific DARPins that inhibit substrate catalysis. Such DARPins might be of significant interest as diagnostic tools to identify BoNT subtypes in clinical samples. DARPin-F5 binds with high affinity to LC/A1 To assess the binding affinity and the kinetic parameters of the interaction of DARPin-F5 with LC/A1 and LC/A3, we performed surface plasmon resonance (SPR) experiments. To this aim, biotin-labelled LC/A1 and LC/A3 were immobilized on streptavidin chips and different concentrations of DARPin-F5 were used as analyte. For the analysis of the interaction of LC/A1 and LC/A3 with DARPin-F5 (Fig. 4 , Supplementary Figure S3), a 1:1 Langmuir-binding model was applied. Because of the slow dissociation rate, kinetic titration was used for the analysis of the binding of DARPin-F5 to LC/A1. We obtained a K d value of 2.38x10 − 10 M for the binding of DARPin-F5 to LC/A1 (Fig. 4 a). In contrast, the interaction of LC/A3 with DARPin-F5 was significantly weaker with a K d of 1.68x10 − 6 M (Fig. 4 b, Supplementary Figure S3), which is consistent with the inability of DARPin-F5 to block substrate catalysis of this subtype. The corresponding association rate constants k on were 5.64x10 6 and 1.91x10 4 M − 1 s − 1 and dissociation rate constants k off of 1.32x10 − 3 and 3.20x10 − 2 s − 1 were obtained for the interaction between DARPin-F5 and LC/A1 and LC/A3, respectively. The slow k off value observed for the interaction between LC/A1 and DARPin-F5 explains the strong binding affinity of the inhibitor to the catalytic domain of the subtype. DARPin-F5 increases BoNT/A1 activity in neuronal cells and muscle tissue We next tested the inhibition potential of DARPin-F5 on BoNT/A1 activity in cells and muscle tissue. Mouse cerebellar granule neurons were incubated with either BoNT/A1 alone or BoNT/A1 preincubated with DARPin-F5. Increasing concentrations of BoNT/A1 were used while the DARPin-F5 concentration was kept constant. Cells were lysed and the SNAP25 content was estimated with an antibody that recognizes both the full-length and the BoNT/A1-cleaved forms of SNAP25. Unexpectedly, we found that DARPin-F5 increased the catalytic activity of BoNT/A1 in neurons (Fig. 5 a). At the lowest toxin concentration, virtually no SNAP25 cleavage occurred while in the presence of DARPin-F5, approximately 50% of SNAP25 was cleaved. We further confirmed the increase in BoNT/A1 activity mediated by DARPin-F5 using the mouse phrenic nerve (MPN) hemidiaphragm assay, an ex vivo muscle paralysis model that represents the standard method to assay the neuroparalytic activity of BoNTs at the neuromuscular junction. In this experimental set up, BoNTs induce a decrease in the twitch capability of the diaphragmatic muscle by exerting their metalloprotease activity on the phrenic nerve. This decay is followed over time to evaluate BoNT potency but is also used to determine the inhibitory activity of antitoxins. The effect DARPin-F5 on the paralytic action of BoNT/A1 at the hemidiaphragm was assessed by treating muscles with either 10 pM BoNT/A1 or the DARPin/toxin complex (molar ratio of 10:1 or 25:1). As shown in Fig. 5 b, 250 pM DARPin-F5 was unable to prevent the paralytic action of BoNT/A1. However, as observed in cells, DARPin-F5 accelerated toxin action and reduced the time of the half-paralysis to 40%. In contrast, the DARPin alone had no effect on the muscle under the experimental conditions tested. DARPin-F5 increases BoNT/A1 translocation To understand the molecular mechanism by which DARPin-F5 increases BoNT/A1 activity in neurons and in the MPN hemidiaphragm assay, we first tested whether DARPin binds to the un-reduced full-length toxin. As shown in Supplementary Figure S2, a small shift of the DARPin/BoNT/A1 complex towards a higher molecular weight was observed by size-exclusion chromatography when compared to the toxin alone. This result indicates that DARPin-F5 can bind to un-reduced full-length BoNT/A1 despite the presence of the disulfide bond-anchored belt. We observed that the BoNT/A1-DARPin-F5 interaction still occurs when the complex was transferred to acidic pH (Supplementary Figure S2), indicating that DARPin-F5 binds to the toxin inside the synaptic vesicles. Together with our findings on increased toxin activity in cells and neuromuscular tissue preparations, it is therefore tempting to speculate that DARPin18 enhances LC translocation probably by dislocating the belt, which might result in destabilization of the catalytic domain. Consistent with this hypothesis, it has been previously shown that stabilization of the BoNT-LC by antibodies or nanobodies can inhibit translocation [ 34 , 36 , 49 ]. Reduction of the interchain disulfide bridge mediated by the thioredoxin-thioredoxin reductase system, is a prerequisite for the release of the LC into the cytosol and the subsequent cleavage of SNARE proteins. This process can be blocked by inhibitors of thioredoxin or of its reductase such as Ebselen [ 50 ]. To estimate the rate of LC/a1 translocation on CNGs, we added Ebselen at various time points after the addition of BoNT/A1 preincubated with or without DARPin-F5. As shown in Fig. 5 c, Ebselen was less effective in blocking SNAP25 cleavage when CGNs were treated with BoNT/A1 preincubated with DARPin-F5. The difference in SNAP25 cleavage was already observable after 10 minutes and was even more pronounced after 60 minutes of BoNT/A1-DARPin-F5 incubation. These results clearly indicate that DARPin-F5 increased LC/A1 entry into the neuronal cytosol, which is consistent with a faster and/or more efficient translocation. Furthermore, the findings also indicate that at some step of translocation process, most likely during unfolding of the LC, DARPin-F5 dissociates from the catalytic domain, which is then free to cleave SNAP25. Discussion DARPins are excellent tools for investigating protein structure and function [ 39 ]. In the present study, we generated DARPins selected against the catalytic domain of BoNT/A1 and characterized them by biochemical, biophysical and structural methods together with functional assays in cells and tissues. We identified a DARPin that inhibits BoNT/A1 catalysis in vitro but, unexpectedly, accelerates toxin activity in cells and muscle tissue. The existence of multiple subtypes within certain BoNT serotypes is a great public health concern because they are all likely to cause botulism. Accurate identification of BoNTs in clinical samples is therefore a fundamental public health goal. A common strategy to achieve this aim is to detect the presence of a particular BoNT through its enzymatic activity on a peptide substrate [ 51 ]. However, a substantial disadvantage of this approach is that many subtypes are frequently not detected by such peptide substrates, indicating differences in their interactions with the substrate. Towards understanding the characteristic features of substrate catalysis of other serotypes and subtypes at the molecular level, structural knowledge of additional toxin-substrate complexes beyond the two examples, LC/A1-SNAP25 [ 47 ] and LC/F1-VAMP2 [ 52 ] that are currently available, seems important. Our data demonstrate that we can generate subtype-specific LC/A DARPins (Fig. 3 ). Such subtype-specific DARPins may offer a diagnostic means complementary to activity assays using peptide substrates to detect BoNT-subtype activity in clinical samples. Surprisingly, DARPin-F5 inhibits SNAP25 cleavage in vitro (Fig. 1 ) but increases LC/A1 activity in neurons and muscle tissue (Fig. 5 ). Our current explanation for the increase of BoNT/A1 activity is that the DARPin prevents the binding of the belt to the LC in a manner similar to the SNAP25 substrate. Crystal structures of BoNT/A, B and E demonstrated that the belt wraps around LC in a way almost identical to SNAP25 and VAMP2 [ 20 – 22 ]. This hypothesis is supported by the observation that DARPin-F5 interacts with full-length BoNT/A1 at neutral and acidic pH (Supplementary Figure S2), which would, on the basis of steric hindrance considerations (Fig. 2 c), not be possible without a conformational change of the belt region. The loss of the interaction of the belt with LC/A1 might result in a faster destabilization of the catalytic domain at low pH, which would then result in faster translocation and subsequent substrate cleavage. This hypothesis is consistent with our BoNT/A1 chase experiment with Ebselen, which revealed faster translocation of the toxin in the presence of DARPin-F5 (Fig. 5 c). It is also in agreement with a recent publication proposing that successful translocation requires LC/A1 destabilization and molten-globule formation at acidic pH [ 53 ]. Consistent with this proposal, it has been recently demonstrated that a nanobodies specific for LC/E1 [ 34 ] or LC/A1 3[ 36 ] or a Fab that binds with high affinity to LC/A1 [ 49 ] efficiently inhibited translocation of the toxin’s catalytic domain, indicating that stabilization of LC is critical for translocation. Furthermore, it has been reported that fusion of GFP, which has a stability of ~ 70°C at acidic pH, to the N-terminus of BoNT/D translocated only with very low efficiency when compared to other, less stable cargo proteins [ 54 ]. However, work remains to be done to clarify how chemical or mechanical protein stability affect translocation. Another factor that has been implicated in faster translocation is the conformation of the full-length toxin. The crystal structures of BoNT/A1 and BoNT/B revealed an open linear arrangement of the three domains with no contact between LC and H C [ 20 , 22 ]. The two domains are separated by H N that wraps around LC as the belt and then folds into the elongated helical translocation domain. In contrast, the crystal structure of BoNT/E shows a more compact arrangement of domains [ 21 ]. LC and H C are positioned on the same side of H N with interactions between all domains. An identical organization of domains is also observed in the cryo-EM structures of BoNT/B and BoNT/E [ 23 ]. It has been suggested that the unique domain arrangement of BoNT/E is the likely reason for its faster translocation and onset of action [ 21 ]. It has also been speculated that for successful translocation the open form of BoNT/A and B will need to convert into closed translocation-competent conformation seen in BoNT/E. This hypothesis was confirmed in recent cryo-EM studies on TeNT, in which the open conformation could be switched into a more compact form upon acidification [ 55 ]. The faster translocation of LC/A1 in the presence of the DARPin might therefore possibly be explained by a faster conformational change of BoNT/A1 from the open into the closed compact form upon DARPin binding. Currently, our knowledge on the role of toxin conformation in translocation is limited, which highlights the need for additional studies to investigate the impact of toxin conformation on translocation. BoNT/A1 is sucessfully used as a therapeutic protein for the treatment of a steadily increasing number of neurological and non-neurological disorders disorders such as strabism, chronic migranes or spasticity, as well as cosmetic applications. Despite its low immunogenicity in humans, treatment with BoNT/A1 can result in some cases in the generation of antibodies against the toxin that prevents further application [ 56 ]. A way to prevent the generation of antibodies against the toxin would be treatment using lower BoNT/A1 doses. To improve applications, pharmaceutical companies are searching for ways to increase BoNT/A1 activity [ 57 ]. These efforts range from site-directed mutagenesis to the supplement of toxin with various additives, including sugars and peptides. Our findings offer the possibility to develop an efficient excipient based on DARPin technology (Fig. 5 ). Taken together, our findings might be of high relevance for the future application of BoNT/A1 (and other BoNTs), which already represents the most frequently used therapeutic protein. Materials And Methods Ribosome display selection of DARPins binding BoNT/A1/LC To generate DARPin binders, biotinylated LC/A1 was immobilized on either MyOne T1 streptavidin-coated beads (Pierce) or Sera-Mag neutravidin-coated beads (GE), depending on the particular selection round. Ribosome display selections were performed essentially as described [ 41 ], using a semi-automatic KingFisher Flex MTP96 well platform. The library includes N3C-DARPins with the original randomization strategy as reported [ 58 ] but includes a stabilized C-cap [ 39 , 59 , 60 ]. Additionally, the library is a mixture of DARPins with randomized and non-randomized N- and C- terminal caps, respectively [ 39 , 61 ] and successively enriched pools were cloned as intermediates in a ribosome display-specific vector. Selections were performed over four rounds with decreasing target concentration and increasing washing steps and the third round included a competition with non-biotinylated LC/A1, to enrich for binders with high affinities. Screening of DARPins binding LC/A1 The final enriched pool of cDNA coding for putative DARPin binders was cloned into a bacterial pQE30 derivative vector (Qiagen), containing a T5 lac promoter and lacIq for expression control, as fusion construct with an N-terminal MRGS(H) 6 tag and C-terminal FLAG tag via unique BamHI and HindIII sites. After transformation of E. coli XL1-blue, 380 single DARPin clones selected to bind LC/A1 were expressed in 96-well format by addition of 1 mM IPTG and lysed by addition of B-Per Direct detergent plus Lysozyme and Nuclease (Pierce). After centrifugation these crude extracts were used for initial screening to bind LC/A1 using HTRF. Binding of the FLAG-tagged DARPins to biotinylated LC/A1, was measured using FRET (donor: Streptavidin-Tb cryptate (610SATLB, Cisbio), acceptor: mAb anti FLAG M2-d2 (61FG2DLB, Cisbio). Further HTRF measurement against ‘No Target’ allowed for discrimination of LC/A1-specific hits. Experiments were performed at room temperature in white 384-well Optiplate plates (PerkinElmer) using the Taglite assay buffer (Cisbio) at a final volume of 20 µl per well. FRET signals were recorded after an incubation time of 30 minutes using a Varioskan LUX Multimode Microplate (Thermo Scientific). HTRF ratios were obtained by dividing the acceptor signal (665 nm) by the donor signal (620 nm) and multiplying this value by 10,000 to derive the 665/620 ratio. The background signal was determined by using reagents in the absence of DARPins. From the initial hits, 32 were chosen and DNA sequence determined by Sanger sequencing. 25 DARPins binding to LC/A1 were identified as single clones. These were expressed and IMAC purified for hit validation ELISA and SEC analysis. Purification of DARPins and hit validation For IMAC purification of the identified 25 DARPins binding to LC/A1 were expressed in small-scale deep-well 96-well plates, lysed with Cell-Lytic B (Sigma) and purified over a 96-well IMAC column (HisPur™ Cobalt plates, Thermo Scientific) including washing with high salt (1 M NaCl) and low salt (20 mM NaCl) PBS buffer. Elution was performed with PBS, 400 mM NaCl, 250 mM imidazole. ELISA was performed with IMAC-purified DARPins against the biotinylated target protein at a concentration of 50 nM on neutravidin-coated 384 wells, or neutravidin only as control. Detection of DARPins at a concentration of 50 nM was performed using a mouse-anti-FLAG M2 monoclonal antibody (dilution 1:5000; Sigma, F1804)) as primary and a goat-anti-mouse antibody conjugated to an alkaline phosphatase (dilution 1:10000; Sigma, A3562) as secondary antibody. After addition of pNPP (para-nitrophenyl phosphate), absorbance at 405 nm was determined after 30 minutes. Signals at A540 nm were subtracted as background correction. Construct design and cloning Codon-optimized synthetic DNA fragments encoding active LC/A1 (UniProtKB entry P0DPI1) variants spanning residues Pro-2 to Gly-421, Pro-2 to Gly-433 and Pro-2 to Lys-448 and active LC/A3 variants (UniProtKB entry D3IV24) comprising residues Pro-2 to Gly-417 and Pro-2 to Lys-444 were cloned into the BamHI/EcoRI site of versions of the expression vectors pET-15b or pET-20b for bacterial production. The pET-15b vector was modified in house to contain a N-terminal MKKHHHHHHGSLVPRGS tag and a different multiple cloning site, and in pET-20b, the pelB leader sequence was replaced by a N-terminal MAHHHHHHGS tag. A codon-optimized synthetic gene fragment encoding residues Met-146 to Gly-204 of human SNAP25 (UniProtKB entry P60880) was cloned into the BamHI/EcoRI site of pHisTrx2, a pET-based expression vector containing an N-terminal 6xHis-tagged thioredoxin A (TrxA) fusion protein [ 62 ]. For biotin-labelled full-length active LC/A1 and LC/A3, an N-terminal Avi-tag (GLNDIFEAQKIEWHE) was introduced via PCR and subsequently the DNA fragments were cloned into the BamHI/XhoI site of the pRSFDuet-1 vector. The active mutants LC/A1-Glu171Asp and LC/A3-Asp171Glu were generated from plasmids encoding the full-length wild-type domains using a modified Quikchange method based on the protocol of Zheng et al. [ 63 ]. All DNA constructs were sequence-verified (Eurofins). All recombinant proteins mentioned in the text contain an N-terminal 6xHis tag unless otherwise stated. Protein expression and purification Protein expression and purification were performed as described previously [ 64 ]. Proteins were expressed in E. coli strain BL21(DE3) (NEB). Bacteria were cultured at 37°C in LB medium containing appropriate antibiotics for selection until an OD 600 of 0.6 was reached. The temperature was then lowered to 18°C, expression was induced with 1 mM IPTG, and incubation continued at 18°C for ~ 16 hours. The cells were harvested by centrifugation (4000 g , 4°C, 15 min) and stored at − 80°C until further use. The His-tagged proteins were purified by Ni-NTA affinity chromatography (GE Healthcare) using a buffer containing 50 mM Tris, pH 7.5, 400 mM NaCl, and 20 mM imidazole. After the washing step with 10 CV (column volumes), the proteins were eluted with a high-imidazole buffer (50 mM Tris, pH 7.5, 400 mM NaCl, and 400 mM imidazole). Pooled fractions of eluted protein were subjected to size exclusion chromatography on a Superdex-200 columm (GE Healthcare) in a buffer containing 20 mM HEPES, pH 7.5, and 150 mM NaCl. For complex formation, LCs and DARPins were combined in a 1:1 ratio, co-purified by size exclusion chromatography (Superdex 200, GE Healthcare) and the pooled fractions concentrated to 8–15 mg/ml for crystallization experiments. The Avi-tagged catalytic domains were expressed according to the protocol from Avidity Avitag™ Technology. Briefly, Avi-tagged LC/A1 and LC/A3 were co-expressed in E . coli strain BL21(DE3) (NEB) with the IPTG-inducible biotin ligase BirA. Bacteria were cultured in TYH media supplemented with 10 µg/ml chloramphenicol, 50 µg/ml kanamycin and 20% glucose. When OD 600 of 0.6 was reached, a biotin solution to a final concentration of 50 µM final was added, protein expression was induced with 1 mM IPTG, and incubation continued for 3 hours at 37°C. Cells were harvested by centrifugation and the Avi-tagged proteins were purified as described above. Sample purity and identity were assessed by SDS-PAGE (Bio-Rad) analysis and Western Blot (Bio-Rad). Protein concentration was estimated by UV at 280 nm and proteins were aliquoted and flash frozen in liquid nitrogen and stored at -80°C until further use. Protease activity assay Experiments were carried out in a 20 µl reaction volume in TBS-150 (20 mM Tris-HCl pH 7.4, 100 mM NaCl). 20 µM of recombinant LCs were incubated with the selected DARPins at different molar ratios (DARPin/toxin ratio of 50:1; 25:1 and 5:1) for 1 hour on ice. 50 µM of purified Trx-SNAP25 was then added to the mixture and incubation continued for 1 h at 37°C. The enzymatic reaction was stopped by adding SDS-PAGE loading buffer and heating for 5 minutes. Samples were subjected to AnyKD gradient SDS-PAGE (Bio-Rad) and gels were stained with Coomassie Blue. The inhibitory effect of the selected DARPin-F5 on LC/A1 and on full-length BoNT/A1 was tested also at molar ratio 2.5:1 (DARPin/toxin). Prior to incubation with DARPin-F5, 1 µM BoNT/A1 was reduced with 10 mM DTT for 30 min at 37°C. Crystallization and structure determination Full-length and truncated LC/A1 variants in complex with DARPin-F5 were concentrated to 8–15 mg/ml and set up for crystallization at 20°C using the sitting-drop vapor diffusion method. Proteins were mixed with the mother liquor in a volume ratio of 1:1 and 2:1. Crystals were only obtained for LC/A1 (residue Pro-2 to Gly-421) in complex with DARPin-F5 in 0.1 M HEPES pH 7.5 containing 28% w/v of jeffamine ED-2003. Crystals typically appeared within 3 days and grew to their maximum size within 1 to 2 weeks. A dataset was collected from single, cryo-cooled crystals diffracting to a resolution of 2.5 Å at beamline PXIII (Swiss Light Source, Villigen, Switzerland) equipped with an EIGER 16M high-resolution detector (Dectris). Raw data were processed and scaled with XDS [ 65 ]. The structure of the LC/A1-DARPin-F5 complex was solved by molecular replacement using the LC/A1-SNAP25 structure (PDB code 1XTG) as a search model [ 47 ]. The structure was built and refined using PHENIX. Manual adjustments of the model were done using COOT [ 66 ]. Crystallographic data and statistics are presented in Supplementary Table S1. The figures were generated with PyMOL (Schrödinger, LLC, New York). Surface Plasmon Resonance (SPR) The binding kinetics of the LC/A1-DARPin-F5 interaction were determined with a kinetic titration [ 67 ] approach by surface plasmon resonance spectroscopy (SPR) on a ProteON XPR36 instrument (BioRad). A NLC sensor chip (BioRad) was used, and all experiments were performed in 10 mM HEPES pH 7.0, 150 mM NaCl, 0.005% Tween-20. Two ligand channels were coated each with 800 RU of in vivo biotinylated LC/A1. Five increasing concentrations of 0.06, 0.19, 0.55, 1.67 and 5 nM of DARPin-F5 were injected consecutively onto the interaction surface in duplicates for 300 s at a flow rate of 80 µL/min, followed by a dissociation phase of 5 min, after which the next higher concentration was injected. The dissociation phase of the highest concentration was set to 2 hours. For data analysis, the measured signals were double-referenced using the ProteOn manager software (Version 3.1.0.6) and fitted to a kinetic titration model using the BiaEvaluation software (Version 4.1.). The binding kinetics of the LC/A3-DARPin-F5 interaction were determined using an OpenSPR instrument (Nicoya). A Streptavidin sensor chip (Nicoya) was used, and the binding experiments were performed in the same buffer that was used for LC/A1. Following equilibration, the two ligand channels were coated each with 400–600 RU of in vivo biotinylated LC/A3 (or LC/A1 as a positive control). Four increasing concentrations of 0.2, 1, 3 and 5 µM of DARPin-F5 were injected as described above, followed by a short dissociation phase, after which the next higher concentration was injected. The measured signals were analyzed with the Tracedrawer Software (Ridgeview Instruments AB) to obtain K d , k on , and k off using a kinetic evaluation assuming a 1:1 binding interaction. Neuronal cell culture and intoxication assay Primary cultures of rat cerebellar granule neurons (CGNs) were prepared from 6 to 8 days-old rats as previously described [ 68 ]. Briefly, cerebella were isolated, mechanically dissociated and trypsinized in the presence of DNase I. Cells were then collected and plated onto 24-well plates pre-coated with poly-L-lysine (50 µg/mL) at a cell density of 4 × 10 5 cells per well. Cultures were maintained at 37°C, 5% CO 2 , 95% humidity in BME (Basal Medium Eagle, Life Technologies) supplemented with 10% fetal bovine serum, 25 mM KCl, 2 mM glutamine and 50 µg/mL gentamicin (hereafter indicated as complete culture medium). To arrest the growth of non-neuronal cells, 10 µM of cytosine arabinoside was added to the complete culture medium 18–24 h after plating. CGNs at 6 days in vitro (DIV) were treated with 0.1, 0.01, 0.005 nM of BoNT/A1, alone or pre-incubated with 3 nM of DARPin in HBS buffer for 2 hours at 4°C. The incubation was prolonged on for 12 or 24 hours at 37°C. To monitor the rate of LC translocation, CGNs were treated with 10 nM of BoNT/A1, alone or pre-incubated with 3 µM of DARPin-F5 in HBS buffer for 2 hours at 4°C. Subsequently, the mix was diluted in complete culture medium supplemented with 60 mM KCl and CGNs were treated with the mix for 5 minutes. After the indicated time points 30 µM of Ebselen was added in fresh medium and the incubation was carried on for 12 hours at 37°C. The specific proteolytic activity against SNAP25 was evaluated using immunoblotting and imaging with specific antibodies. Immunoblotting Cells were directly lysed with Laemmli sample buffer supplemented with protease inhibitors (Roche). Cell lysates were loaded onto NuPage 4–12% Bis-Tris gels (Life Technologies) and separated by electrophoresis in MOPS buffer. Proteins were transferred onto Protran nitrocellulose membranes (Whatman) and saturated for 1 hour in PBS-T (PBS, 0.1% Tween 20) supplemented with 5% non-fatty milk. Anti-SNAP-25 (SMI-81, 1:10000) was from Biologend; anti-VAMP-2 (104211, 1:2000) was from Synaptic System; anti-SNAP-25 BoNT/A-cleaved (1:5000) was home made and used as previously described [ 69 ]; anti-Syntaxin-1A/1B (1:2000) was home made and used as previously described [ 70 ]. Incubation with indicated primary antibodies was performed overnight at 4°C. The membranes were then washed three times with PBS-T and incubated with appropriate HRP-conjugated secondary antibodies (Anti mouse 1:5000 and Anti rabbit& mouse Flourescent labelled 1:2000) for 1 h. Membranes were washed three times with PBS and proteins revealed with an Uvitec gel doc system (Uvitec Cambridge). Mouse phrenic nerve (MPN) hemidiaphragm assay The experiments were performed in accordance with the European Communities Council Directive n° 2010/63/UE. The isolated mouse diaphragms were prepared from CD-1 mice weighing about 20–25 g and halved into two contralateral hemi-diaphragms still innervated with their own phrenic nerve. Muscles were mounted into two chambers filled with 4 ml of oxygenated (95% O 2 , 5% CO 2 ) solution (139 mM NaCl, 12 mM NaHCO 3 , 4 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 1 mM KH 2 PO 4 and 11 mM glucose, pH 7.4). The two phrenic nerves were stimulated via two ring platinum electrodes with supramaximal stimuli of 3 V amplitude and 0. 1 ms pulse duration, with a frequency of 0.1 Hz. Muscle contraction was monitored with an isometric transducer (Harvard Apparatus); data were recorded and analyzed via an iWORX 118 system with Labscribe software (Harvard Apparatus). DARPin-toxin mixtures were prepared at three different molar ratios of 10:1, 25:1 and 50:1 and added directly to the oxygenated solution of one muscle, and the same volume of toxin at 10 pM final concentration was added alone to the contralateral one for direct comparison. The twitch was monitored until complete paralysis was achieved. Graphs show muscle twitching capability over time, reported as a percentage with respect to the initial value obtained before toxin addition. Declarations ACKNOWLEDGMENTS The PXI beamline staff (Swiss Light Source, Villigen, Switzerland) is acknowledged for their support during the diffraction experiments. We thank Dr. Paola Caccin (University of Padova, Italy) for technical assistance in conducting the MPN hemidiaphragm assays and analysis. Prof. Ornella Rossetto (University of Padova, Italy) is acknowledged for the provision of antibodies. We thank Dr. Birgit Dreier for coordinating the work towards the end of the selection project. 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Zurich","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Plückthun","suffix":""},{"id":169123025,"identity":"9821fc7a-66d6-428b-988e-d9f57bec79fe","order_by":8,"name":"Xiaodan Li","email":"","orcid":"https://orcid.org/0000-0001-8647-7317","institution":"Paul Scherrer Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaodan","middleName":"","lastName":"Li","suffix":""},{"id":169123026,"identity":"f238ddd2-0c64-4227-a743-e81a26f2aa19","order_by":9,"name":"Marco Pirazzini","email":"","orcid":"","institution":"University of Padova","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Pirazzini","suffix":""},{"id":169123027,"identity":"c65672b6-f6f9-49c4-83f2-e8bb231cbd88","order_by":10,"name":"Richard Kammerer","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYBAC+2YGNiDFzGBwACrCD8QHwAgHMDgM1WLYABWRbCCk5QBUizGSCAMDXi3HmZ89+LnHmsGM/XTqho97bPKNb7c/PMBQcwePX9jMDXuepTPY8ORuuznjWZrltjtngBYde4ZTix0zD5sEz4HDDDYMudtuAxkGZjdyGA4wNhzGqcUYqEXyD1CLGf/bbbf/HPhvYDwj/QFeLYbNPGzSIFuMJYC2MBw4YGAgkWCAV4vBYTYzaZkD6TyGM95uu9lzINlAAuSXhGN4tJw//EzyzQFrOYPzudtu/DhgZ8A/u/3xhw81uLXAAA+CKQHECQQ1oAAJ0pSPglEwCkbB8AcAIE9faaqtI6QAAAAASUVORK5CYII=","orcid":"","institution":"Paul Scherrer Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Richard","middleName":"","lastName":"Kammerer","suffix":""}],"badges":[],"createdAt":"2022-12-24 11:15:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2411408/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2411408/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-023-44102-4","type":"published","date":"2023-12-18T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":31839313,"identity":"9115d2ff-9192-4395-b528-8fb8c9351a4d","added_by":"auto","created_at":"2023-01-20 05:01:14","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":48276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of enzymatic activity of BoNT/A1 and recombinant LC/A1 by DARPin-F5. \u003c/strong\u003e(a) LC/A1 was incubated with DARPin-F5 (DARPin/toxin ratio of 2.5:1). After incubation, the recombinant substrate Trx-SNAP25 was added to the mixture and the catalytic activity of LC/A1 was abolished (lane 4). (b) The same experimental conditions were applied to reduced full-length BoNT/A1. In both experiments, samples were analyzed by SDS-PAGE and Coomassie Blue staining. The position of the BoNT/A1 heavy chain (HC/A1), BoNT/A1 light chain (LC/A1), recombinant full-length LC/A1 (residues P2-K448), cleaved and uncleaved Trx-SNAP25 substrate and DARPin-F5 are indicated by arrows\u003cem\u003e.\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eThe positions of marker proteins are indicated.\u003c/p\u003e","description":"","filename":"Slide1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2411408/v1/b21c99a81aad883d4cee79a6.jpg"},{"id":31838796,"identity":"d5c0e57e-b32e-454b-8723-f4dbcb41148e","added_by":"auto","created_at":"2023-01-20 04:53:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":116334,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCrystal structure of the LC/A1-DARPin-F5 complex. \u003c/strong\u003e(a) Cartoon representation of the complex structure (PDB code 8HKH). LC/A1 is shown in cyan and DARPin-F5 in orange. The zinc ion is shown as a grey sphere. Superimposition of the LC/A1-DARPin-F5 complex structure to (b) the LC/A1-SNAP25 complex structure (PDB code 1XTG) and (c) the structure of the BoNT/A1 holotoxin (PDB code 3BTA) reveals that DARPin-F5 is interfering with substrate and belt binding, respectively. α- and β-exosites are encircled. The SNAP25 peptide shown in blue. The belt region of BoNT/A1 is shown in red. N- and C-termini of all proteins are indicated in the colors used for their representation.\u003c/p\u003e","description":"","filename":"Slide2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2411408/v1/8bbf581582c2ecdb26a7b0ed.jpg"},{"id":31838797,"identity":"b5b61ecd-f887-4946-907e-592943a56393","added_by":"auto","created_at":"2023-01-20 04:53:14","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":84667,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubtype-specific inhibition of BoNT/A by DARPin-F5.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representation of the most prominent interaction between Glu-171 of LC/A1 (in orange) and Lys-91, Arg-25, and Tyr-50 of DARPin-F5. b) Conservation of the DARPin-F5 binding regions of LC/A1 (Thr-125 to Asn-132 and Ile-161 to Leu-175) and the corresponding regions of the other seven BoNT/A subtypes. Glu-171 (indicated by an arrow) is present only in BoNT/A1 and A6 while in the other subtypes an aspartate is found at the corresponding position. The other two interacting residues, LC/A1 Lys-128 and Asp-131, are identical in all subtypes. Clustal Omega was used for generating the sequence alignment. *, identical residue; :, strong similarity. Loop 170 is indicated by the cyan line. (c) The impact of DARPin-F5 on the catalysis of wild-type LC/A1 and LC/A3 (residues Pro-2 to Lys-444) and their respective mutants (LC/A1-Glu171Asp and LC/A3-Asp171Glu). DARPin-F5 was not able to inhibit substrate cleavage by LC/A3 (gel 2, lane 2) and LC/A1-Glu171Asp (gel 3, lane 2). In contrast, DARPin-F5 completely blocked the protease activity of LC/A1 (gel 1, lane 2) and LC/A3-Asp173Glu (gel 4, lane 2). Recombinant wild-type and mutant LCs, cleaved, uncleaved substrate and DARPin-F5 are indicated by arrows.\u003c/p\u003e","description":"","filename":"Slide3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2411408/v1/93dd03296985a8ed58005587.jpg"},{"id":31839397,"identity":"280987e5-bfdb-45b7-9329-e1f18baac406","added_by":"auto","created_at":"2023-01-20 05:09:14","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":52354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding affinity and kinetic parameters of the interaction of DARPin-F5 with LC/A1 and LC/A3. \u003c/strong\u003e(a) SPR sensorgram of DARPin-F5 binding to immobilized LC/A1 (black) overlaid with a fit of a 1:1 binding model (red line) using the BiaEvaluation software (Version 4.1). Increasing DARPin-F5 concentrations from 0.06 to 5 nM were applied. (b) Table summarizing the k\u003csub\u003eon\u003c/sub\u003e, k\u003csub\u003eoff\u003c/sub\u003e and K\u003csub\u003ed \u003c/sub\u003evalues of DARPin-F5 binding to LC/A1 and LC/A3.\u003c/p\u003e","description":"","filename":"Slide4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2411408/v1/d046ecfa231de87cb5924399.jpg"},{"id":31838799,"identity":"0942c212-b911-4bb3-9791-3c29976a0749","added_by":"auto","created_at":"2023-01-20 04:53:14","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":53855,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIncrease of DARPin-mediated enzymatic activity of BoNT/A1 in cells and muscle tissue.\u003c/strong\u003e (a) Cerebellar granule neurons (CGNs) were incubated with BoNT/A1 alone (lanes 2, 4, 6, 8) of the BoNT/A1-DARPin-F5 complex (lanes 3, 5, 7, 9) at decreasing BoNT/A1 concentrations of 1, 0.1, 0.01 and 0.001 nM and a constant DARPin-F5 concentration of 3 nM. Lane 1, CGN control without toxin. Cells were lysed and the SNARE content was estimated using the indicated antibodies: SMI81 (anti-SNAP25) recognizes both the full-length (red) and the cleaved form of SNAP25 (green) and BoNT/A-cleaved recognizes only BoNT/A1-truncated SNAP25. An antibody against intact form of VAMP2 was used as loading control. SNARE proteins recognized by the antibodies are indicated by arrows. (b) The neurotoxicity of 10 pM of BoNT/A1 in the presence of 250 pM DARPin-F5 was assessed in the mouse phrenic nerve (MPN) hemidiaphragm assay. Data are represented as mean values and error bars indicate the standard deviation obtained from at least three independent experiments. (c) DARPin-F5 increases the translocation of BoNT/A1. A clear difference in substrate cleavage indicating different rates of translocation was observed in Ebselen-treated cells with or without the DARPin already after 10 minutes. Cells were lysed and the SNAP25 content estimated as in (a). Syntaxin-1A (STX-1A) was used as loading control.\u003c/p\u003e","description":"","filename":"Slide5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2411408/v1/3fb1221d5051c959c676131b.jpg"},{"id":48439135,"identity":"84c29003-b6f8-43ac-ab8e-da20814d9c5f","added_by":"auto","created_at":"2023-12-19 08:08:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":794394,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2411408/v1/b672137e-aee9-4ad6-ad9e-b96246754a59.pdf"},{"id":31838802,"identity":"99b11341-5e8c-438f-8fc2-e66df4e9b2b7","added_by":"auto","created_at":"2023-01-20 04:53:14","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5391210,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Figures and Tables\u003c/p\u003e","description":"","filename":"SupplementaryMaterials.pptx","url":"https://assets-eu.researchsquare.com/files/rs-2411408/v1/ab21d529ea4bf855d4fea325.pptx"},{"id":31839314,"identity":"4f5bdb4d-71ea-4d56-a253-6522613c711e","added_by":"auto","created_at":"2023-01-20 05:01:14","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2664034,"visible":true,"origin":"","legend":"\u003cp\u003eReporting Summary\u003c/p\u003e","description":"","filename":"NCOMMS2252937rs.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2411408/v1/d9a5e38e93af8ded5d5656dc.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A DARPin Increases the Catalytic Activity of Botulinum Neurotoxin A1","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBotulinum neurotoxins (BoNTs) produced by anaerobic bacteria of the genus \u003cem\u003eClostridium\u003c/em\u003e are the most poisonous bacterial protein toxins known [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. BoNT intoxication in vertebrates causes botulism, a potentially life-threatening neuroparalytic syndrome. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Therefore the toxins represent potential biological weapons. Despite their toxicity and as a result of their characteristics, including biological effectiveness and long persistence of action in patients, BoNTs are nowadays the most widely used therapeutic proteins in various human neurological and non-neurological disorders [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Furthermore, they are used in cosmetic applications. Traditionally, BoNTs are classified as seven serologically distinct proteins, referred to as BoNT/A through BoNT/G, however, their genetic variability is further increased by the existence of more than 40 subtypes within serotypes A, B, E and F [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Although many subtypes have not yet been well characterized, it was shown that some differ from the other proteins within a serotype with respect to their catalytic properties, substrate specificity, duration of action, and efficiency to enter neuronal cells [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Because they could outperform conventionally used BoNTs with respect to their biological activities, it appears important to characterize all BoNT subtypes. Furthermore, there exist mosaic toxins between serotypes C and D [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and partially between A and F [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Recently, several BoNT-like sequences have been discovered in single bacterial strains whose occurrence is of great concern, because it indicates that \u003cem\u003eClostridium botulinum\u003c/em\u003e is able to horizontally transfer BoNT genes to other bacterial species [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBoNTs are produced as single precursors that share a common domain organization [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. They are processed into mature toxins by cleavage into a\u0026thinsp;~\u0026thinsp;50 kDa light chain (LC) and a\u0026thinsp;~\u0026thinsp;100 kDa heavy chain (HC) that remain connected through an interchain disulfide bond.\u003c/p\u003e \u003cp\u003eThe LC is a zinc-dependent endopeptidase that specifically cleaves members of the soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) family of proteins, which are key components of the vesicular fusion machinery within presynaptic nerve terminals. BoNT/A and E cleave synaptosomal-associated protein 25 (SNAP25), and BoNT/B, D, F and G cut vesicle-associated membrane protein (VAMP). BoNT/C can cleave two substrates, SNAP25 and syntaxin [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. SNARE cleavage blocks acetylcholine release at the neuromuscular junction, leading to a flaccid paralysis of muscles [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe HC consists of a 50 kDa N-terminal translocation domain (H\u003csub\u003eN\u003c/sub\u003e) and a C-terminal receptor-binding domain (H\u003csub\u003eC\u003c/sub\u003e) of similar size. A dual interaction mode involving two different receptors seems to be required for the neuro-specific binding of BoNTs [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Most BoNTs bind a polysialoganglioside (PSG) and synaptotagmin (Syt) or synaptic vesicle glycoprotein 2 (SV2) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUpon binding to neuronal cell receptors, BoNTs are endocytosed into synaptic vesicles [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The low pH and the lipid environment within the synaptic vesicle is believed to initiate conformational changes in H\u003csub\u003eN\u003c/sub\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Amongst those, exposure of a hydrophobic peptide through a viral-fusion-peptide-like pH-dependent molecular switch seems to initiate the first step of membrane insertion of BoNT/A1 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Subsequent conformational changes are thought to lead to the formation of an ion-conductive transmembrane channel through which the LC is translocated across the membrane. Once inside the reducing intracellular environment of the cytosol, the disulfide bond is reduced, resulting in release of the protease.\u003c/p\u003e \u003cp\u003eThe existence of multiple BoNT and BoNT-like toxin molecules represents a great public health threat, because they can potentially all cause botulism or be engaged as bioweapons. As result of this public health concern, multiple strategies have been developed for the treatment and prevention of botulism [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Like the toxoid-based tetanus vaccination against the structurally related Tetanus neurotoxin (TeNT), vaccination using BoNT toxoid efficiently protects against botulism [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Although different next-generation vaccines are being developed to replace the discontinued toxoid vaccine [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], vaccination is seldom used because botulism is a rare disease with less than 200 cases reported per year [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. More importantly, vaccination would have a negative impact on the therapeutic benefits of BoNTs for medical applications. As a result, passive immunotherapy using polyclonal antibody preparations and sera, individual monoclonal antibodies and monoclonal antibody combinations are used for the medical treatment of botulism [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In addition, alternative strategies such as the application of camelid single-chain antibodies are also being developed [\u003cspan additionalcitationids=\"CR34 CR35 CR36 CR37\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA class of proteins whose usefulness as BoNT-inhibiting agents has not yet been explored are the DARPins (designed ankyrin repeat proteins) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. DARPins are small, engineered antibody mimetics derived from synthetic libraries of consensus ankyrin proteins that are very specific and show very high affinity to the target protein. They are successfully used in various research, diagnostic and therapeutic applications. Considerable benefits of DARPins are their small size of 14 or 18 kDa (4 or 5 ankyrin repeats of which two or three carry a randomized surface and are flanked by N-capping and C-capping repeats) and their high stability and solubility. In addition, DARPins can be generated rapidly and cost-efficiently in a soluble form and high yields in the cytoplasm of \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eBiomolecules like nanobodies and DARPins might contribute significantly to the development and of next-generation biomolecular antidotes against botulism. However, since they typically recognize conformational epitopes, nanobodies and DARPins also represent invaluable tools to probe the structure and function of BoNTs.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the impact of DARPins selected against LC/A1 on toxin activity using biochemical and biophysical methods, X-ray crystallography and functional assays performed \u003cem\u003ein vitro\u003c/em\u003e and in cells and tissue.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGeneration and characterization of DARPins selected against LC/A1\u003c/h2\u003e \u003cp\u003eDARPins were selected over four rounds of ribosome display [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] using a semi-automated 96 well platform against full-length LC/A1 (amino-acid residues Pro-2 to Lys-448). In total for each selection 380 single clones were screened using a 384 well-based HTRF assay. From the initial hits, 32 were sequenced. For the selection against full-length LC/A1, 25 specific and unique clones were identified. These were expressed and IMAC purified. Binding was confirmed using ELISA.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDARPin-F5 inhibits the catalytic activity of LC/A1\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e\u003c/p\u003e \u003cp\u003eBecause its C-terminus contributes to catalysis, the full-length LC represents the relevant target for BoNT/A1 inhibitor studies [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In a first step, we were searching for DARPins that inhibit the protease activity of full-length LC/A1. To this end, we incubated each of the 25 selected DARPins individually at different ratios with LC/A1 and monitored the cleavage of the recombinant human SNAP-25 substrate peptide spanning amino-acid residues Met-141 to Gly-204. We identified six DARPins that completely or partially inhibited the proteolytic activity of BoNT LC/A1. Among the candidates, DARPin 008-829-2386-F5 (named DARPin-F5 for short) was able to completely inhibit substrate cleavage at a DARPin/toxin molar ratio of 2.5:1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) while the remaining five DARPins showed partial inhibition of LC/A1 enzymatic activity to varying degrees (data not shown).\u003c/p\u003e \u003cp\u003eBecause the C-terminus of the catalytic domain has been implicated in enzyme/substrate interactions [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], we also tested the ability of DARPin-F5 to prevent the cleavage of the SNAP25 peptide substrate using two C-terminally truncated LC/A1 variants (residues Pro-2 to Gly-421 and Pro-2 to Gly-433). DARPin-F5 also completely inhibited proteolytic activity of both truncated LC/A1 variants (not shown), indicating that DARPin-F5 doesn\u0026rsquo;t bind to the C-terminus of the catalytic domain.\u003c/p\u003e \u003cp\u003eIn full-length BoNT/A1 the catalytic domain is surrounded by the belt region, which is considered a pseudo-substrate that prevents LC/A1 protease activity before its delivery into the neuronal cytosol [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Although it is unknown if LC and HC remain bound to each other after reduction of the connecting disulfide bridge, reduced full-length BoNT/A1 efficiently cleaves SNAP25. As observed for LC/A1, DARPin-F5 was found to also completely inhibit reduced full-length BoNT/A1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCrystal structure of the LC/A1-DARPin-F5 complex\u003c/h2\u003e \u003cp\u003eTo understand at the molecular level how DARPin-F5 inhibits the catalytic activity of LC/A1, we next focused on determining the crystal structure of the complex. Attempts to crystallize DARPin-F5 with the full-length catalytic domain or a slightly shorter LC/A1 variant (amino-acid residues Pro-2 to Gly-433) were not successful, probably because these variants are too flexible for crystallization and prone to aggregation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Therefore, we focused on an even more truncated LC/A1 variant spanning amino-acid residues Pro-2 to Gly-421, which was previously used to solve the crystal structure of a LC/A1-SNAP25 toxin/substrate complex [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Crystals suitable for structural work were obtained within 1\u0026ndash;2 weeks and the structure of the LC/A1-DARPin-F5 complex was solved at a resolution of 2.5 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Supplementary Table S1; PDB code 8HKH). The crystal structure contains two toxin/DARPin complexes per asymmetric unit (rmsd value of 0.2 \u0026Aring; for 510 Cα atoms of both AC and BD complexes). In the following, we analyzed the AC complex.\u003c/p\u003e \u003cp\u003eIn botulinum neurotoxins, SNAP25 wraps around the catalytic domain in a manner very similar to the belt [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The binding interface includes the α-exosite and the β-exosite, two major structural motifs that bind the ~\u0026thinsp;60 residue-long substrate remote from the catalytic site. It has been shown that the exosites play important roles for substrate binding and specificity [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The crystal structure of the complex reveals that DARPin-F5 binds to a substrate-binding site between the α- and β-exosites of LC/A1, far away from the active site of the catalytic domain. The DARPin therefore inhibits SNAP25 hydrolysis by preventing substrate binding. Consistent with this observation, it has been shown that nanobodies preventing the binding of the substrate also show potent inhibition of LC/A1 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAll BoNT-LCs share a globular domain fold in which a conserved substrate-binding groove extends from the catalytic site around the enzyme [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Loops 50, 170, 250, and 370 form the boundaries of this large cleft on the enzyme surface that in the neurotoxin holostructure is occupied by the belt. Superimposition of the LC/A1-DARPin-F5 complex structure with the structure of LC/A1 bound to SNAP25 (PDB code 1XTG) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] shows that DARPin-F5 binds to the region of loop 170, thereby preventing the interaction of SNAP25 with this site (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The belt binds to LC/A1 in the full-length toxin in a manner that is very similar to the interaction of LC/A1 with the substrate [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Accordingly, DARPin could also interfere with the binding of the belt as shown in the superimposition of the LC/A1-DARPin-F5 complex structure with the structure of BoNT/A1 holotoxin (PBD 3BTA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eThe most prominent feature seen in the complex structure is a network of interactions involving Glu-171 of LC/A1 and three residues from different loops of the DARPin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). These interactions include a salt bridge that is formed between Glu-171 of LC/A1 and Lys-91 of the DARPin, respectively. In addition, Arg-25, and the phenolic hydroxy group of Tyr-50 of DARPin-F5 form hydrogen bonds with the backbone oxygen and the carbonyl group of Glu-171, respectively. Notably, Arg-25 is conserved amongst DARPins. Based on structural considerations, Glu-171 of LC/A1 seems to be a key residue for the interaction with DARPin-F5, although it is not directly involved in the interaction with the SNAP25 substrate.\u003c/p\u003e \u003cp\u003eMoreover, two additional residues from LC/A1, Lys-128 and Asp-131, are also involved in the complex formation with DARPin-F5. The interactions seen at the interface are summarized in Supplementary Table S2.\u003c/p\u003e \u003cp\u003eIn addition, the 6xHis tag present in the DARPin (chains C and D) also interacts with the LC/A1 (chains B and A, respectively) of the other complex (Supplementary Figure S1). Residues His-11 to Gly-13 form a short β-strand that interacts in an antiparallel manner with the β-strand formed by residues Gly-255 to Phe-260. As a result of this interaction, the N-terminus (His-7 to His-10) points deep into the active site and extends towards the catalytic zinc. This second interaction is unlikely to contribute to the inhibition of LC/A1, because there is no evidence of tetrameric complexes in solution (Supplementary Figure S2). The interaction is therefore probably needed to establish the crystal packing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDARPin-F5 is a subtype-specific inhibitor of BoNT/A catalysis\u003c/h2\u003e \u003cp\u003eAs shown in the alignment of Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, the DARPin-binding region is well conserved amongst BoNT/A subtypes. Glu-171 of LC/A1 is a conserved Asp in all other subtypes except BoNT/A6. The other two interacting residues, LC/A1 Lys-128 and Asp-131, are identical in all subtypes. The conservation of the DARPin-binding region suggests that DARPin-F5 might inhibit the catalytic activity of all eight BoNT/A subtypes. To test this hypothesis, we assesed whether DARPin-F5 is able to block the enzymatic activity of LC/A3, a potentially very attractive subtype for medical applications such as pain relief and orthopedics where a shorter onset and duration of action are required [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. We found that DARPin-F5 was not able to inhibit SNAP25 catalysis by LC/A3. Therefore, we next focused on the role of Glu-171 in the inhibition of SNAP25 catalysis and generated LC/A1 and LC/A3 mutants, in which Glu-171 and Asp-171 were exchanged between subtypes. First, we tested the activity of the mutants: LC/A1-Glu171Asp and LC/A3-Asp171Glu cleaved SNAP25 with the same efficiency as the respective wild-type proteins. DARPin-F5 completely blocked SNAP25 cleavage by LC/A3-Asp171Glu (DARPin-F5/toxin molar ratio of 5:1 or 10:1) but did not inhibit substrate catalysis of LC/A1 Glu171Asp (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), demonstrating the importance of this amino-acid residue for DARPin-F5-mediated inhibition of proteolysis.\u003c/p\u003e \u003cp\u003eTaken together, our findings demonstrate that we can generate BoNT/A subtype-specific DARPins that inhibit substrate catalysis. Such DARPins might be of significant interest as diagnostic tools to identify BoNT subtypes in clinical samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDARPin-F5 binds with high affinity to LC/A1\u003c/h2\u003e \u003cp\u003eTo assess the binding affinity and the kinetic parameters of the interaction of DARPin-F5 with LC/A1 and LC/A3, we performed surface plasmon resonance (SPR) experiments. To this aim, biotin-labelled LC/A1 and LC/A3 were immobilized on streptavidin chips and different concentrations of DARPin-F5 were used as analyte. For the analysis of the interaction of LC/A1 and LC/A3 with DARPin-F5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Figure S3), a 1:1 Langmuir-binding model was applied. Because of the slow dissociation rate, kinetic titration was used for the analysis of the binding of DARPin-F5 to LC/A1. We obtained a K\u003csub\u003ed\u003c/sub\u003e value of 2.38x10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e M for the binding of DARPin-F5 to LC/A1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In contrast, the interaction of LC/A3 with DARPin-F5 was significantly weaker with a K\u003csub\u003ed\u003c/sub\u003e of 1.68x10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e M (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, Supplementary Figure S3), which is consistent with the inability of DARPin-F5 to block substrate catalysis of this subtype. The corresponding association rate constants k\u003csub\u003eon\u003c/sub\u003e were 5.64x10\u003csup\u003e6\u003c/sup\u003e and 1.91x10\u003csup\u003e4\u003c/sup\u003e M\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and dissociation rate constants k\u003csub\u003eoff\u003c/sub\u003e of 1.32x10\u003csup\u003e\u0026minus;\u0026thinsp;3\u003c/sup\u003e and 3.20x10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were obtained for the interaction between DARPin-F5 and LC/A1 and LC/A3, respectively. The slow k\u003csub\u003eoff\u003c/sub\u003e value observed for the interaction between LC/A1 and DARPin-F5 explains the strong binding affinity of the inhibitor to the catalytic domain of the subtype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDARPin-F5 increases BoNT/A1 activity in neuronal cells and muscle tissue\u003c/h2\u003e \u003cp\u003eWe next tested the inhibition potential of DARPin-F5 on BoNT/A1 activity in cells and muscle tissue. Mouse cerebellar granule neurons were incubated with either BoNT/A1 alone or BoNT/A1 preincubated with DARPin-F5. Increasing concentrations of BoNT/A1 were used while the DARPin-F5 concentration was kept constant. Cells were lysed and the SNAP25 content was estimated with an antibody that recognizes both the full-length and the BoNT/A1-cleaved forms of SNAP25. Unexpectedly, we found that DARPin-F5 increased the catalytic activity of BoNT/A1 in neurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). At the lowest toxin concentration, virtually no SNAP25 cleavage occurred while in the presence of DARPin-F5, approximately 50% of SNAP25 was cleaved.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further confirmed the increase in BoNT/A1 activity mediated by DARPin-F5 using the mouse phrenic nerve (MPN) hemidiaphragm assay, an \u003cem\u003eex vivo\u003c/em\u003e muscle paralysis model that represents the standard method to assay the neuroparalytic activity of BoNTs at the neuromuscular junction. In this experimental set up, BoNTs induce a decrease in the twitch capability of the diaphragmatic muscle by exerting their metalloprotease activity on the phrenic nerve. This decay is followed over time to evaluate BoNT potency but is also used to determine the inhibitory activity of antitoxins.\u003c/p\u003e \u003cp\u003eThe effect DARPin-F5 on the paralytic action of BoNT/A1 at the hemidiaphragm was assessed by treating muscles with either 10 pM BoNT/A1 or the DARPin/toxin complex (molar ratio of 10:1 or 25:1). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, 250 pM DARPin-F5 was unable to prevent the paralytic action of BoNT/A1. However, as observed in cells, DARPin-F5 accelerated toxin action and reduced the time of the half-paralysis to 40%. In contrast, the DARPin alone had no effect on the muscle under the experimental conditions tested.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDARPin-F5 increases BoNT/A1 translocation\u003c/h2\u003e \u003cp\u003eTo understand the molecular mechanism by which DARPin-F5 increases BoNT/A1 activity in neurons and in the MPN hemidiaphragm assay, we first tested whether DARPin binds to the un-reduced full-length toxin. As shown in Supplementary Figure S2, a small shift of the DARPin/BoNT/A1 complex towards a higher molecular weight was observed by size-exclusion chromatography when compared to the toxin alone. This result indicates that DARPin-F5 can bind to un-reduced full-length BoNT/A1 despite the presence of the disulfide bond-anchored belt. We observed that the BoNT/A1-DARPin-F5 interaction still occurs when the complex was transferred to acidic pH (Supplementary Figure S2), indicating that DARPin-F5 binds to the toxin inside the synaptic vesicles. Together with our findings on increased toxin activity in cells and neuromuscular tissue preparations, it is therefore tempting to speculate that DARPin18 enhances LC translocation probably by dislocating the belt, which might result in destabilization of the catalytic domain. Consistent with this hypothesis, it has been previously shown that stabilization of the BoNT-LC by antibodies or nanobodies can inhibit translocation [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eReduction of the interchain disulfide bridge mediated by the thioredoxin-thioredoxin reductase system, is a prerequisite for the release of the LC into the cytosol and the subsequent cleavage of SNARE proteins. This process can be blocked by inhibitors of thioredoxin or of its reductase such as Ebselen [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. To estimate the rate of LC/a1 translocation on CNGs, we added Ebselen at various time points after the addition of BoNT/A1 preincubated with or without DARPin-F5.\u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, Ebselen was less effective in blocking SNAP25 cleavage when CGNs were treated with BoNT/A1 preincubated with DARPin-F5. The difference in SNAP25 cleavage was already observable after 10 minutes and was even more pronounced after 60 minutes of BoNT/A1-DARPin-F5 incubation. These results clearly indicate that DARPin-F5 increased LC/A1 entry into the neuronal cytosol, which is consistent with a faster and/or more efficient translocation. Furthermore, the findings also indicate that at some step of translocation process, most likely during unfolding of the LC, DARPin-F5 dissociates from the catalytic domain, which is then free to cleave SNAP25.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDARPins are excellent tools for investigating protein structure and function [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In the present study, we generated DARPins selected against the catalytic domain of BoNT/A1 and characterized them by biochemical, biophysical and structural methods together with functional assays in cells and tissues. We identified a DARPin that inhibits BoNT/A1 catalysis \u003cem\u003ein vitro\u003c/em\u003e but, unexpectedly, accelerates toxin activity in cells and muscle tissue.\u003c/p\u003e \u003cp\u003eThe existence of multiple subtypes within certain BoNT serotypes is a great public health concern because they are all likely to cause botulism. Accurate identification of BoNTs in clinical samples is therefore a fundamental public health goal. A common strategy to achieve this aim is to detect the presence of a particular BoNT through its enzymatic activity on a peptide substrate [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. However, a substantial disadvantage of this approach is that many subtypes are frequently not detected by such peptide substrates, indicating differences in their interactions with the substrate. Towards understanding the characteristic features of substrate catalysis of other serotypes and subtypes at the molecular level, structural knowledge of additional toxin-substrate complexes beyond the two examples, LC/A1-SNAP25 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and LC/F1-VAMP2 [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] that are currently available, seems important.\u003c/p\u003e \u003cp\u003eOur data demonstrate that we can generate subtype-specific LC/A DARPins (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Such subtype-specific DARPins may offer a diagnostic means complementary to activity assays using peptide substrates to detect BoNT-subtype activity in clinical samples.\u003c/p\u003e \u003cp\u003eSurprisingly, DARPin-F5 inhibits SNAP25 cleavage \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) but increases LC/A1 activity in neurons and muscle tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Our current explanation for the increase of BoNT/A1 activity is that the DARPin prevents the binding of the belt to the LC in a manner similar to the SNAP25 substrate. Crystal structures of BoNT/A, B and E demonstrated that the belt wraps around LC in a way almost identical to SNAP25 and VAMP2 [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This hypothesis is supported by the observation that DARPin-F5 interacts with full-length BoNT/A1 at neutral and acidic pH (Supplementary Figure S2), which would, on the basis of steric hindrance considerations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), not be possible without a conformational change of the belt region. The loss of the interaction of the belt with LC/A1 might result in a faster destabilization of the catalytic domain at low pH, which would then result in faster translocation and subsequent substrate cleavage. This hypothesis is consistent with our BoNT/A1 chase experiment with Ebselen, which revealed faster translocation of the toxin in the presence of DARPin-F5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). It is also in agreement with a recent publication proposing that successful translocation requires LC/A1 destabilization and molten-globule formation at acidic pH [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Consistent with this proposal, it has been recently demonstrated that a nanobodies specific for LC/E1 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] or LC/A1 3[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] or a Fab that binds with high affinity to LC/A1 [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] efficiently inhibited translocation of the toxin\u0026rsquo;s catalytic domain, indicating that stabilization of LC is critical for translocation. Furthermore, it has been reported that fusion of GFP, which has a stability of ~\u0026thinsp;70\u0026deg;C at acidic pH, to the N-terminus of BoNT/D translocated only with very low efficiency when compared to other, less stable cargo proteins [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. However, work remains to be done to clarify how chemical or mechanical protein stability affect translocation.\u003c/p\u003e \u003cp\u003eAnother factor that has been implicated in faster translocation is the conformation of the full-length toxin. The crystal structures of BoNT/A1 and BoNT/B revealed an open linear arrangement of the three domains with no contact between LC and H\u003csub\u003eC\u003c/sub\u003e [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The two domains are separated by H\u003csub\u003eN\u003c/sub\u003e that wraps around LC as the belt and then folds into the elongated helical translocation domain. In contrast, the crystal structure of BoNT/E shows a more compact arrangement of domains [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. LC and H\u003csub\u003eC\u003c/sub\u003e are positioned on the same side of H\u003csub\u003eN\u003c/sub\u003e with interactions between all domains. An identical organization of domains is also observed in the cryo-EM structures of BoNT/B and BoNT/E [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It has been suggested that the unique domain arrangement of BoNT/E is the likely reason for its faster translocation and onset of action [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It has also been speculated that for successful translocation the open form of BoNT/A and B will need to convert into closed translocation-competent conformation seen in BoNT/E. This hypothesis was confirmed in recent cryo-EM studies on TeNT, in which the open conformation could be switched into a more compact form upon acidification [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The faster translocation of LC/A1 in the presence of the DARPin might therefore possibly be explained by a faster conformational change of BoNT/A1 from the open into the closed compact form upon DARPin binding.\u003c/p\u003e \u003cp\u003eCurrently, our knowledge on the role of toxin conformation in translocation is limited, which highlights the need for additional studies to investigate the impact of toxin conformation on translocation.\u003c/p\u003e \u003cp\u003eBoNT/A1 is sucessfully used as a therapeutic protein for the treatment of a steadily increasing number of neurological and non-neurological disorders disorders such as strabism, chronic migranes or spasticity, as well as cosmetic applications. Despite its low immunogenicity in humans, treatment with BoNT/A1 can result in some cases in the generation of antibodies against the toxin that prevents further application [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. A way to prevent the generation of antibodies against the toxin would be treatment using lower BoNT/A1 doses. To improve applications, pharmaceutical companies are searching for ways to increase BoNT/A1 activity [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. These efforts range from site-directed mutagenesis to the supplement of toxin with various additives, including sugars and peptides. Our findings offer the possibility to develop an efficient excipient based on DARPin technology (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaken together, our findings might be of high relevance for the future application of BoNT/A1 (and other BoNTs), which already represents the most frequently used therapeutic protein.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRibosome display selection of DARPins binding BoNT/A1/LC\u003c/h2\u003e \u003cp\u003eTo generate DARPin binders, biotinylated LC/A1 was immobilized on either MyOne T1 streptavidin-coated beads (Pierce) or Sera-Mag neutravidin-coated beads (GE), depending on the particular selection round. Ribosome display selections were performed essentially as described [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], using a semi-automatic KingFisher Flex MTP96 well platform.\u003c/p\u003e \u003cp\u003eThe library includes N3C-DARPins with the original randomization strategy as reported [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] but includes a stabilized C-cap [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Additionally, the library is a mixture of DARPins with randomized and non-randomized N- and C- terminal caps, respectively [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e] and successively enriched pools were cloned as intermediates in a ribosome display-specific vector. Selections were performed over four rounds with decreasing target concentration and increasing washing steps and the third round included a competition with non-biotinylated LC/A1, to enrich for binders with high affinities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eScreening of DARPins binding LC/A1\u003c/h2\u003e \u003cp\u003eThe final enriched pool of cDNA coding for putative DARPin binders was cloned into a bacterial pQE30 derivative vector (Qiagen), containing a T5 lac promoter and lacIq for expression control, as fusion construct with an N-terminal MRGS(H)\u003csub\u003e6\u003c/sub\u003e tag and C-terminal FLAG tag via unique BamHI and HindIII sites. After transformation of \u003cem\u003eE. coli\u003c/em\u003e XL1-blue, 380 single DARPin clones selected to bind LC/A1 were expressed in 96-well format by addition of 1 mM IPTG and lysed by addition of B-Per Direct detergent plus Lysozyme and Nuclease (Pierce). After centrifugation these crude extracts were used for initial screening to bind LC/A1 using HTRF. Binding of the FLAG-tagged DARPins to biotinylated LC/A1, was measured using FRET (donor: Streptavidin-Tb cryptate (610SATLB, Cisbio), acceptor: mAb anti FLAG M2-d2 (61FG2DLB, Cisbio). Further HTRF measurement against \u0026lsquo;No Target\u0026rsquo; allowed for discrimination of LC/A1-specific hits. Experiments were performed at room temperature in white 384-well Optiplate plates (PerkinElmer) using the Taglite assay buffer (Cisbio) at a final volume of 20 \u0026micro;l per well. FRET signals were recorded after an incubation time of 30 minutes using a Varioskan LUX Multimode Microplate (Thermo Scientific). HTRF ratios were obtained by dividing the acceptor signal (665 nm) by the donor signal (620 nm) and multiplying this value by 10,000 to derive the 665/620 ratio. The background signal was determined by using reagents in the absence of DARPins.\u003c/p\u003e \u003cp\u003eFrom the initial hits, 32 were chosen and DNA sequence determined by Sanger sequencing. 25 DARPins binding to LC/A1 were identified as single clones. These were expressed and IMAC purified for hit validation ELISA and SEC analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePurification of DARPins and hit validation\u003c/h2\u003e \u003cp\u003eFor IMAC purification of the identified 25 DARPins binding to LC/A1 were expressed in small-scale deep-well 96-well plates, lysed with Cell-Lytic B (Sigma) and purified over a 96-well IMAC column (HisPur\u0026trade; Cobalt plates, Thermo Scientific) including washing with high salt (1 M NaCl) and low salt (20 mM NaCl) PBS buffer. Elution was performed with PBS, 400 mM NaCl, 250 mM imidazole.\u003c/p\u003e \u003cp\u003eELISA was performed with IMAC-purified DARPins against the biotinylated target protein at a concentration of 50 nM on neutravidin-coated 384 wells, or neutravidin only as control. Detection of DARPins at a concentration of 50 nM was performed using a mouse-anti-FLAG M2 monoclonal antibody (dilution 1:5000; Sigma, F1804)) as primary and a goat-anti-mouse antibody conjugated to an alkaline phosphatase (dilution 1:10000; Sigma, A3562) as secondary antibody. After addition of pNPP (para-nitrophenyl phosphate), absorbance at 405 nm was determined after 30 minutes. Signals at A540 nm were subtracted as background correction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eConstruct design and cloning\u003c/h2\u003e \u003cp\u003eCodon-optimized synthetic DNA fragments encoding active LC/A1 (UniProtKB entry P0DPI1) variants spanning residues Pro-2 to Gly-421, Pro-2 to Gly-433 and Pro-2 to Lys-448 and active LC/A3 variants (UniProtKB entry D3IV24) comprising residues Pro-2 to Gly-417 and Pro-2 to Lys-444 were cloned into the BamHI/EcoRI site of versions of the expression vectors pET-15b or pET-20b for bacterial production. The pET-15b vector was modified in house to contain a N-terminal MKKHHHHHHGSLVPRGS tag and a different multiple cloning site, and in pET-20b, the pelB leader sequence was replaced by a N-terminal MAHHHHHHGS tag. A codon-optimized synthetic gene fragment encoding residues Met-146 to Gly-204 of human SNAP25 (UniProtKB entry P60880) was cloned into the BamHI/EcoRI site of pHisTrx2, a pET-based expression vector containing an N-terminal 6xHis-tagged thioredoxin A (TrxA) fusion protein [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor biotin-labelled full-length active LC/A1 and LC/A3, an N-terminal Avi-tag (GLNDIFEAQKIEWHE) was introduced via PCR and subsequently the DNA fragments were cloned into the BamHI/XhoI site of the pRSFDuet-1 vector. The active mutants LC/A1-Glu171Asp and LC/A3-Asp171Glu were generated from plasmids encoding the full-length wild-type domains using a modified Quikchange method based on the protocol of Zheng et al. [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. All DNA constructs were sequence-verified (Eurofins). All recombinant proteins mentioned in the text contain an N-terminal 6xHis tag unless otherwise stated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eProtein expression and purification\u003c/h2\u003e \u003cp\u003eProtein expression and purification were performed as described previously [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Proteins were expressed in \u003cem\u003eE. coli\u003c/em\u003e strain BL21(DE3) (NEB). Bacteria were cultured at 37\u0026deg;C in LB medium containing appropriate antibiotics for selection until an OD\u003csub\u003e600\u003c/sub\u003e of 0.6 was reached. The temperature was then lowered to 18\u0026deg;C, expression was induced with 1 mM IPTG, and incubation continued at 18\u0026deg;C for ~\u0026thinsp;16 hours. The cells were harvested by centrifugation (4000 \u003cem\u003eg\u003c/em\u003e, 4\u0026deg;C, 15 min) and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further use.\u003c/p\u003e \u003cp\u003eThe His-tagged proteins were purified by Ni-NTA affinity chromatography (GE Healthcare) using a buffer containing 50 mM Tris, pH 7.5, 400 mM NaCl, and 20 mM imidazole. After the washing step with 10 CV (column volumes), the proteins were eluted with a high-imidazole buffer (50 mM Tris, pH 7.5, 400 mM NaCl, and 400 mM imidazole). Pooled fractions of eluted protein were subjected to size exclusion chromatography on a Superdex-200 columm (GE Healthcare) in a buffer containing 20 mM HEPES, pH 7.5, and 150 mM NaCl. For complex formation, LCs and DARPins were combined in a 1:1 ratio, co-purified by size exclusion chromatography (Superdex 200, GE Healthcare) and the pooled fractions concentrated to 8\u0026ndash;15 mg/ml for crystallization experiments.\u003c/p\u003e \u003cp\u003eThe Avi-tagged catalytic domains were expressed according to the protocol from Avidity Avitag\u0026trade; Technology. Briefly, Avi-tagged LC/A1 and LC/A3 were co-expressed in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e strain BL21(DE3) (NEB) with the IPTG-inducible biotin ligase BirA. Bacteria were cultured in TYH media supplemented with 10 \u0026micro;g/ml chloramphenicol, 50 \u0026micro;g/ml kanamycin and 20% glucose. When OD\u003csub\u003e600\u003c/sub\u003e of 0.6 was reached, a biotin solution to a final concentration of 50 \u0026micro;M final was added, protein expression was induced with 1 mM IPTG, and incubation continued for 3 hours at 37\u0026deg;C. Cells were harvested by centrifugation and the Avi-tagged proteins were purified as described above. Sample purity and identity were assessed by SDS-PAGE (Bio-Rad) analysis and Western Blot (Bio-Rad). Protein concentration was estimated by UV at 280 nm and proteins were aliquoted and flash frozen in liquid nitrogen and stored at -80\u0026deg;C until further use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eProtease activity assay\u003c/h2\u003e \u003cp\u003eExperiments were carried out in a 20 \u0026micro;l reaction volume in TBS-150 (20 mM Tris-HCl pH 7.4, 100 mM NaCl). 20 \u0026micro;M of recombinant LCs were incubated with the selected DARPins at different molar ratios (DARPin/toxin ratio of 50:1; 25:1 and 5:1) for 1 hour on ice. 50 \u0026micro;M of purified Trx-SNAP25 was then added to the mixture and incubation continued for 1 h at 37\u0026deg;C. The enzymatic reaction was stopped by adding SDS-PAGE loading buffer and heating for 5 minutes. Samples were subjected to AnyKD gradient SDS-PAGE (Bio-Rad) and gels were stained with Coomassie Blue. The inhibitory effect of the selected DARPin-F5 on LC/A1 and on full-length BoNT/A1 was tested also at molar ratio 2.5:1 (DARPin/toxin). Prior to incubation with DARPin-F5, 1 \u0026micro;M BoNT/A1 was reduced with 10 mM DTT for 30 min at 37\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCrystallization and structure determination\u003c/h2\u003e \u003cp\u003eFull-length and truncated LC/A1 variants in complex with DARPin-F5 were concentrated to 8\u0026ndash;15 mg/ml and set up for crystallization at 20\u0026deg;C using the sitting-drop vapor diffusion method. Proteins were mixed with the mother liquor in a volume ratio of 1:1 and 2:1. Crystals were only obtained for LC/A1 (residue Pro-2 to Gly-421) in complex with DARPin-F5 in 0.1 M HEPES pH 7.5 containing 28% w/v of jeffamine ED-2003. Crystals typically appeared within 3 days and grew to their maximum size within 1 to 2 weeks. A dataset was collected from single, cryo-cooled crystals diffracting to a resolution of 2.5 \u0026Aring; at beamline PXIII (Swiss Light Source, Villigen, Switzerland) equipped with an EIGER 16M high-resolution detector (Dectris). Raw data were processed and scaled with XDS [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. The structure of the LC/A1-DARPin-F5 complex was solved by molecular replacement using the LC/A1-SNAP25 structure (PDB code 1XTG) as a search model [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The structure was built and refined using PHENIX. Manual adjustments of the model were done using COOT [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Crystallographic data and statistics are presented in Supplementary Table S1. The figures were generated with PyMOL (Schr\u0026ouml;dinger, LLC, New York).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSurface Plasmon Resonance (SPR)\u003c/h2\u003e \u003cp\u003eThe binding kinetics of the LC/A1-DARPin-F5 interaction were determined with a kinetic titration [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e] approach by surface plasmon resonance spectroscopy (SPR) on a ProteON XPR36 instrument (BioRad). A NLC sensor chip (BioRad) was used, and all experiments were performed in 10 mM HEPES pH 7.0, 150 mM NaCl, 0.005% Tween-20. Two ligand channels were coated each with 800 RU of \u003cem\u003ein vivo\u003c/em\u003e biotinylated LC/A1. Five increasing concentrations of 0.06, 0.19, 0.55, 1.67 and 5 nM of DARPin-F5 were injected consecutively onto the interaction surface in duplicates for 300 s at a flow rate of 80 \u0026micro;L/min, followed by a dissociation phase of 5 min, after which the next higher concentration was injected. The dissociation phase of the highest concentration was set to 2 hours. For data analysis, the measured signals were double-referenced using the ProteOn manager software (Version 3.1.0.6) and fitted to a kinetic titration model using the BiaEvaluation software (Version 4.1.).\u003c/p\u003e \u003cp\u003eThe binding kinetics of the LC/A3-DARPin-F5 interaction were determined using an OpenSPR instrument (Nicoya). A Streptavidin sensor chip (Nicoya) was used, and the binding experiments were performed in the same buffer that was used for LC/A1. Following equilibration, the two ligand channels were coated each with 400\u0026ndash;600 RU of \u003cem\u003ein vivo\u003c/em\u003e biotinylated LC/A3 (or LC/A1 as a positive control). Four increasing concentrations of 0.2, 1, 3 and 5 \u0026micro;M of DARPin-F5 were injected as described above, followed by a short dissociation phase, after which the next higher concentration was injected. The measured signals were analyzed with the Tracedrawer Software (Ridgeview Instruments AB) to obtain K\u003csub\u003ed\u003c/sub\u003e, k\u003csub\u003eon\u003c/sub\u003e, and k\u003csub\u003eoff\u003c/sub\u003e using a kinetic evaluation assuming a 1:1 binding interaction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eNeuronal cell culture and intoxication assay\u003c/h2\u003e \u003cp\u003ePrimary cultures of rat cerebellar granule neurons (CGNs) were prepared from 6 to 8 days-old rats as previously described [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Briefly, cerebella were isolated, mechanically dissociated and trypsinized in the presence of DNase I. Cells were then collected and plated onto 24-well plates pre-coated with poly-L-lysine (50 \u0026micro;g/mL) at a cell density of 4 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well. Cultures were maintained at 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e, 95% humidity in BME (Basal Medium Eagle, Life Technologies) supplemented with 10% fetal bovine serum, 25 mM KCl, 2 mM glutamine and 50 \u0026micro;g/mL gentamicin (hereafter indicated as complete culture medium). To arrest the growth of non-neuronal cells, 10 \u0026micro;M of cytosine arabinoside was added to the complete culture medium 18\u0026ndash;24 h after plating. CGNs at 6 days \u003cem\u003ein vitro\u003c/em\u003e (DIV) were treated with 0.1, 0.01, 0.005 nM of BoNT/A1, alone or pre-incubated with 3 nM of DARPin in HBS buffer for 2 hours at 4\u0026deg;C. The incubation was prolonged on for 12 or 24 hours at 37\u0026deg;C. To monitor the rate of LC translocation, CGNs were treated with 10 nM of BoNT/A1, alone or pre-incubated with 3 \u0026micro;M of DARPin-F5 in HBS buffer for 2 hours at 4\u0026deg;C. Subsequently, the mix was diluted in complete culture medium supplemented with 60 mM KCl and CGNs were treated with the mix for 5 minutes. After the indicated time points 30 \u0026micro;M of Ebselen was added in fresh medium and the incubation was carried on for 12 hours at 37\u0026deg;C. The specific proteolytic activity against SNAP25 was evaluated using immunoblotting and imaging with specific antibodies.\u003c/p\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003eImmunoblotting\u003c/h2\u003e \u003cp\u003eCells were directly lysed with Laemmli sample buffer supplemented with protease inhibitors (Roche). Cell lysates were loaded onto NuPage 4\u0026ndash;12% Bis-Tris gels (Life Technologies) and separated by electrophoresis in MOPS buffer. Proteins were transferred onto Protran nitrocellulose membranes (Whatman) and saturated for 1 hour in PBS-T (PBS, 0.1% Tween 20) supplemented with 5% non-fatty milk. Anti-SNAP-25 (SMI-81, 1:10000) was from Biologend; anti-VAMP-2 (104211, 1:2000) was from Synaptic System; anti-SNAP-25 BoNT/A-cleaved (1:5000) was home made and used as previously described [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]; anti-Syntaxin-1A/1B (1:2000) was home made and used as previously described [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Incubation with indicated primary antibodies was performed overnight at 4\u0026deg;C. The membranes were then washed three times with PBS-T and incubated with appropriate HRP-conjugated secondary antibodies (Anti mouse 1:5000 and Anti rabbit\u0026amp; mouse Flourescent labelled 1:2000) for 1 h. Membranes were washed three times with PBS and proteins revealed with an Uvitec gel doc system (Uvitec Cambridge).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMouse phrenic nerve (MPN) hemidiaphragm assay\u003c/h2\u003e \u003cp\u003eThe experiments were performed in accordance with the European Communities Council Directive n\u0026deg; 2010/63/UE. The isolated mouse diaphragms were prepared from CD-1 mice weighing about 20\u0026ndash;25 g and halved into two contralateral hemi-diaphragms still innervated with their own phrenic nerve. Muscles were mounted into two chambers filled with 4 ml of oxygenated (95% O\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e) solution (139 mM NaCl, 12 mM NaHCO\u003csub\u003e3\u003c/sub\u003e, 4 mM KCl, 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and 11 mM glucose, pH 7.4). The two phrenic nerves were stimulated via two ring platinum electrodes with supramaximal stimuli of 3 V amplitude and 0. 1 ms pulse duration, with a frequency of 0.1 Hz. Muscle contraction was monitored with an isometric transducer (Harvard Apparatus); data were recorded and analyzed via an iWORX 118 system with Labscribe software (Harvard Apparatus). DARPin-toxin mixtures were prepared at three different molar ratios of 10:1, 25:1 and 50:1 and added directly to the oxygenated solution of one muscle, and the same volume of toxin at 10 pM final concentration was added alone to the contralateral one for direct comparison. The twitch was monitored until complete paralysis was achieved. Graphs show muscle twitching capability over time, reported as a percentage with respect to the initial value obtained before toxin addition.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;PXI\u0026nbsp;beamline staff\u0026nbsp;(Swiss Light Source, Villigen, Switzerland)\u0026nbsp;is acknowledged for their support during the diffraction experiments. We thank Dr. Paola Caccin (University of Padova, Italy) for technical assistance in conducting the MPN hemidiaphragm assays and analysis. Prof. Ornella Rossetto (University of Padova, Italy) is acknowledged for the provision of antibodies. We thank Dr. Birgit Dreier for coordinating the work towards the end of the selection project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.A.K., J.S., A.P. X.L. and M.P. designed the research; O.L., Y.W. S.F., T.R., J. M. and G.Z. carried out the research; O.L., Y.W., M.P, and R.A.K. analyzed the data; O.L. and R.A.K. wrote the manuscript with input from the other authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING AND ADDITIONAL INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants 31003A_163449 and 31003A_170028 of the Swiss National Science Foundation to R.A.K. and X.L., respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRossetto, O., M. Pirazzini, and C. Montecucco, \u003cem\u003eBotulinum neurotoxins: genetic, structural and mechanistic insights\u003c/em\u003e. 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Toxins (Basel), 2015. \u003cb\u003e7\u003c/b\u003e(12): p.\u0026nbsp;5322\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZanetti, G., et al., \u003cem\u003eBotulinum neurotoxin C mutants reveal different effects of syntaxin or SNAP-25 proteolysis on neuromuscular transmission\u003c/em\u003e. PLoS Pathog, 2017. \u003cb\u003e13\u003c/b\u003e(8): p.\u0026nbsp;e1006567.\u003c/span\u003e\u003c/li\u003e\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-2411408/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2411408/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, we characterized Designed Ankyrin Repat Proteins (DARPins) as investigative tools to probe botulinum neurotoxin (BoNT) function and as potential antidotes for botulism. We selected DARPins against the catalytic domain of BoNT/A1 and characterized them by biochemical, biophysical and structural studies in combination with functional assays in cultured neurons and muscle tissue. We identified DARPin-F5 that completely blocks SNAP25 substrate cleavage by BoNT/A1 \u003cem\u003ein vitro\u003c/em\u003e. X-ray crystallography revealed that DARPin-F5 inhibits BoNT/A1 activity by interacting with a substrate-binding region between the α- and β-exosite. This DARPin blocked substrate cleavage of BoNT/A1 but not of BoNT/A3, indicating that DARPin-F5 is a subtype-specific inhibitor. We found that BoNT/A1 Glu-171 plays a critical role in the interaction with DARPin-F5 and its mutation to Asp, the residue found in BoNT/A3, resulted in a loss of inhibition of substrate cleavage by reducing DARPin affinity from pM to \u0026micro;M. In contrast to the \u003cem\u003ein vitro\u003c/em\u003e results, DARPin-F5 increased BoNT/A1 activity in primary neurons. This result was confirmed by the mouse phrenic nerve hemidiaphragm assay, demonstrating faster paralysis in the presence of the DARPin. We show by functional studies in neuronal cells that DARPin-F5 increases translocation of the toxin. Our findings could have important implications for the identification of BoNTs in clinical samples as well as the development of excipients that allow BoNT treatment at a lower dosage and thereby prevent the generation of antibodies against the toxin.\u003c/p\u003e","manuscriptTitle":"A DARPin Increases the Catalytic Activity of Botulinum Neurotoxin A1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-01-20 04:53:09","doi":"10.21203/rs.3.rs-2411408/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":"292d669a-1752-4603-a209-b259b82c95e0","owner":[],"postedDate":"January 20th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":18543273,"name":"Biological sciences/Structural biology/X-ray crystallography"},{"id":18543274,"name":"Biological sciences/Biochemistry/Enzymes/Proteases"},{"id":18543275,"name":"Biological sciences/Cell biology/Protein transport"}],"tags":[],"updatedAt":"2023-12-19T08:07:55+00:00","versionOfRecord":{"articleIdentity":"rs-2411408","link":"https://doi.org/10.1038/s41467-023-44102-4","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2023-12-18 05:00:00","publishedOnDateReadable":"December 18th, 2023"},"versionCreatedAt":"2023-01-20 04:53:09","video":"","vorDoi":"10.1038/s41467-023-44102-4","vorDoiUrl":"https://doi.org/10.1038/s41467-023-44102-4","workflowStages":[]},"version":"v1","identity":"rs-2411408","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2411408","identity":"rs-2411408","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}