rongsheng jin lab manufacturer

Lam, K. H., Guo, Z., Krez, N., Matsui, T., Perry, K., Weisemann, J., Rummel, A., Bowen, M. E. & Jin, R. A viral-fusion-peptide-like molecular switch drives membrane insertion of botulinum neurotoxin A1. Nat Commun 9, 5367 (2018) doi: 10.1038/s41467-018-07789-4.

Chen, P., Tao, L., Liu, Z., Dong, M. & Jin, R. Structural insight into Wnt signaling inhibition by Clostridium difficile toxin B. FEBS J (2018) doi: 10.1111/febs.14681.

Chen, P., Tao, L., Wang, T., Zhang, J., He, A., Lam, K. H., Liu, Z., He, X., Perry, K., Dong, M*. & Jin, R*. Structural basis for recognition of frizzled proteins by Clostridium difficile toxin B. Science 360, 664-669 (2018) (*corresponding authors) doi: 10.1126/science.aar1999. PMCID: PMC6231499

Lam, K. H., Sikorra, S., Weisemann, J., Maatsch, H., Perry, K., Rummel, A., Binz, T. & Jin, R. Structural and biochemical characterization of the protease domain of the mosaic botulinum neurotoxin type HA. Pathog Dis 76 (2018) doi: 10.1093/femspd/fty044. PMCID: PMC5961070

Silva, D. A., Stewart, L., Lam, K. H., Jin, R. & Baker, D. Structures and disulfide cross-linking of de novo designed therapeutic mini-proteins. FEBS J 285, 1783-1785 (2018) doi: 10.1111/febs.14394. PMCID: PMC6001749

Lam, K. H., Qi, R., Liu, S., Kroh, A., Yao, G., Perry, K., Rummel, A. & Jin, R. The hypothetical protein P47 of Clostridium botulinum E1 strain Beluga has a structural topology similar to bactericidal/permeability-increasing protein. Toxicon 147, 19-26 (2018) doi: 10.1016/j.toxicon.2017.10.012. PMCID: PMC5902665

Chevalier, A., Silva, D.A., Rocklin, G.J., Hicks, D.R., Vergara, R., Murapa, P., Bernard, S.M., Zhang, L., Lam, K.H., Yao, G., Bahl, C.D., Miyashita, S.I., Goreshnik, I., Fuller, J.T., Koday, M.T., Jenkins, C.M., Colvin, T., Carter, L., Bohn, A., Bryan, C.M., Fernández-Velasco, D.A., Stewart, L., Dong, M., Huang, X., Jin, R., Wilson, I.A., Fuller, D.H. & Baker, D. Massively parallel de novo protein design for targeted therapeutics. Nature 550(7674):74-79 (2017) doi: 10.1038/nature23912. PMCID: PMC5802399

Yao, G., Lam, K.H., Weisemann, J., Peng, L., Krez, N., Perry, K., Shoemaker, C.B., Dong, M., Rummel, A. & Jin, R. A camelid single-domain antibody neutralizes botulinum neurotoxin A by blocking host receptor binding. Sci Rep. 7;7(1):7438. (2017) doi: 10.1038/s41598-017-07457-5. PMCID: PMC5547058

Yao, G., Lam, K.H., Perry, K., Weisemann, J., Rummel, A. & Jin, R. Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H. Toxins (Basel) 9, 93 (2017) doi: 10.3390/toxins9030093. PMCID: PMC5371848

Yao, G., Zhang, S., Mahrhold, S., Lam, K. H., Stern, D., Bagramyan, K., Perry, K., Kalkum, M., Rummel, A.*, Dong, M.* & Jin, R.* N-linked glycosylation of SV2 is required for binding and uptake of botulinum neurotoxin A. Nat Struct Mol Biol 23 (7):656-662 (2016) (*corresponding authors) doi: 10.1038/nsmb.3245. PMCID: PMC5033645

Lee, K., Lam, K. H., Kruel, A. M., Mahrhold, S., Perry, K., Cheng, L. W., Rummel, A. & Jin, R. Inhibiting oral intoxication of botulinum neurotoxin A complex by carbohydrate receptor mimics. Toxicon 107, 43-49 (2015) doi: 10.1016/j.toxicon.2015.08.003. PMCID: PMC4658216

Lam, K.H. & Jin, R. Architecture of the botulinum neurotoxin complex: a molecular machine for protection and delivery. Current Opinion in Structural Biology 31:89-95 (2015) doi: 10.1016/j.sbi.2015.03.013. PMCID: PMC4476938

Lam, K.H., Yao, G. & Jin, R. Diverse binding modes, same goal: The receptor recognition mechanism of botulinum neurotoxin. Progress in Biophysics and Molecular Biology 117(2-3):225-31 (2015) doi: 10.1016/j.pbiomolbio.2015.02.004. PMCID: PMC4417461

Lam, T.I., Stanker, L.H., Lee, K., Jin, R. & Cheng, L.W. Translocation of botulinum neurotoxin serotype A and associated proteins across the intestinal epithelia. Cellular Microbiology 17(8):1133-1143 (2015) doi: 10.1111/cmi.12424. PMCID: PMC4610714

Matsui, T.*, Gu, S., Lam, K.H., Carter, L.G., Rummel, A., Mathews, II. & Jin, R.* Structural Basis of the pH-Dependent Assembly of a Botulinum Neurotoxin Complex. J. Mol. Biol. 426(22):3773-3782 (2014) doi: 10.1016/j.jmb.2014.09.009. (*corresponding authors) PMCID: PMC4252799

Lee, K., Zhong, X., Gu, S., Kruel, A.M., Dorner, M.B., Perry, K., Rummel, A., Dong, M. & Jin, R. Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin A complex. Science 344(6190):1405-1410 (2014) doi: 10.1126/science.1253823. PMCID: PMC4164303

Lee, K., Lam, K.H., Kruel, A.M., Perry, K., Rummel, A. and Jin, R. High-resolution crystal structure of HA33 of botulinum neurotoxin type B progenitor toxin complex. Biochem. Biophys. Res. Commun. 446(2):568-573 (2014) doi: 10.1016/j.bbrc.2014.03.008. PMCID: PMC4020412

Yao, Y., Lee, K., Gu, S., Lam, K.H. & Jin, R. Botulinum Neurotoxin A Complex Recognizes Host Carbohydrates through Its Hemagglutinin Component, Toxins (Basel) 6(2):624-635 (2014) doi: 10.3390/toxins6020624. PMCID: PMC3942755

Lee, K., Gu, S., Jin, L., Le, T.T.N., Cheng, L.W., Strotmeier, J., Kruel, A.M., Yao, G., Perry, K., Rummel, A.* & Jin, R.* Structure of a Bimodular Botulinum Neurotoxin Complex Provides Insights into Its Oral Toxicity. PLoS Pathog. 9(10): e1003690 (2013) doi:10.1371/journal.ppat.1003690. (*corresponding authors) PMCID: PMC3795040

Zong, Y. and Jin, R. Structural mechanisms of the agrin-LRP4-MuSK signaling pathway in neuromuscular junction differentiation. Cell. Mol. Life Sci. 70(17):3077-88 (2013) doi: 10.1007/s00018-012-1209-9. PMCID: PMC4627850

Gu, S. and Jin, R. Assembly and function of the botulinum neurotoxin progenitor complex. Curr. Top. Microbiol. Immunol. 364:21-44 (2013) doi: 10.1007/978-3-642-33570-9_2. PMCID: PMC3875173

Gu, S., Rumpel, S., Zhou, J., Strotmeier, J., Bigalke, H., Perry, K., Shoemaker, C.B., Rummel, A. & Jin, R. Botulinum neurotoxin is shielded by NTNHA in an interlocked complex. Science 335(6071):977-81 (2012) doi: 10.1126/science.1214270. PMCID: PMC3545708

Zong, Y., Zhang, B., Gu, S., Lee, K., Zhou, J., Yao, G., Figueiredo, D., Perry, K., Mei, L.* & Jin, R.* Structural basis of neuron-specific regulation of postsynaptic differentiation. Gene & Development 26:247-258 (2012) doi: 10.1101/gad.180885.111. (*corresponding authors) PMCID: PMC3278892

Yao, G., Zong, Y., Gu, S., Zhou, J., Xu, H., Mathews, II. & Jin, R. Crystal structure of the glutamate receptor GluA1 amino-terminal domain. Biochem. J. 438(2):255-63 (2011) doi: 10.1042/BJ20110801. PMCID: PMC3296483

Strotmeier, J., Gu, S., Jutzi, S., Mahrhold, S., Zhou, J., Pich, A., Eichner, T., Bigalke, H., Rummel, A.*, Jin, R.* & Binz, T*. The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites. Mol. Microbiol. 81(1):143-56 (2011) doi: 10.1111/j.1365-2958.2011.07682.x. Epub 2011 Jun 2. (*corresponding authors)

Strotmeier, J., Lee, K., Völker, A.K., Mahrhold, S., Zong, Y., Zeiser, J., Zhou, J., Pich, A., Bigalke, H., Binz, T., Rummel, A.* & Jin, R.* Botulinum neurotoxin serotype D attacks neurons via two carbohydrate-binding sites in a ganglioside-dependent manner. Biochem. J. 431(2):207-16 (2010) (*corresponding authors)

Jin, R.*, Singh, S.K., Gu, S., Furukawa, H., Sobolevsky, A.I., Zhou, J., Jin, Y. & Gouaux E.* Crystal structure and association behavior of the GluR2 amino-terminal domain. EMBO J. 28(12):1812-23 (2009) (*corresponding authors) PMCID: PMC2699365

Kumar, J., Schuck. P., Jin, R. & Mayer, M.L. The N-terminal domain of GluR6-subtype glutamate receptor ion channels. Nat. Struct. Mol. Biol. 16(6):631-8 (2009) PMCID: PMC2729365

Jin, R., Rummel, A., Binz, T. & Brunger, A.T. Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity. Nature 444:1092-5 (2006)

Jin, R., Clark, S., Weeks, A.M., Dudman, J.T., Gouaux, E. & Partin, K.M. Mechanism of positive allosteric modulators acting on AMPA receptors. J. Neurosci. 25(39):9027-36 (2005)

Jin, R., Junutula, J.R., Matern, H.T., Ervin, K.E., Scheller, R.H. & Brunger, A.T. Exo84 and Sec5 are competitive regulatory Sec6/8 effectors to the RalA GTPase. EMBO J. 24:2064-74 (2005)

Jin, R., Bank, T., Mayer, M. L., Traynelis, S. & Gouaux, E. Structural basis for partial agonist action at ionotropic glutamate receptors. Nat. Neurosci. 6(8):803-10 (2003)

Jahid S, Ortega JA, Vuong LM, Acquistapace IM, Hachey SJ, Flesher JL, La Serra MA, Brindani N, La Sala G, Manigrasso J, Arencibia JM, Bertozzi SM, Summa M, Bertorelli R, Armirotti A, Jin R, Liu Z, Chen CF, Edwards R, Hughes CCW, De Vivo M, Ganesan AK. PMID: 35385746; PMCID: PMC9127750.

Chen P, Zeng J, Liu Z, Thaker H, Wang S, Tian S, Zhang J, Tao L, Gutierrez CB, Xing L, Gerhard R, Huang L, Dong M, Jin R. PMID: 34145250; PMCID: PMC8213806.

Chen P, Lam KH, Liu Z, Mindlin FA, Chen B, Gutierrez CB, Huang L, Zhang Y, Hamza T, Feng H, Matsui T, Bowen ME, Perry K, Jin R. PMID: 31308519; PMCID: PMC6684407.

Zheng Liu, Conceptualization, Investigation, Visualization, Writing - original draft,1 Sicai Zhang, Conceptualization, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft, Writing - review & editing,2 Peng Chen, Conceptualization, Methodology,1 Songhai Tian, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing,2 Ji Zeng, Investigation, Resources,2 Kay Perry, Investigation,3 Min Dong, Conceptualization, Funding acquisition, Methodology, Project administration, Supervision, Visualization, Writing - original draft, Writing - review & editing,2 and Rongsheng Jin, Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing1

To understand how these structural differences exemplified by GTDVPI10463 and GTDM68 are related to the evolution of diverse TcdB variants, we carried out structure-based sequence analyses among all known TcdB variants available in DiffBase, which have been classified into 12 subfamilies (B1 to B12) (fig. S8) (18). We found that all these TcdB variants could be classified into two distinct groups based on their sequences in the GTPase-binding areas. Specifically, characteristic Rho-binding residues are conserved not only in other members of the same subfamily of TcdBVPI10463 (B1) but also in members of the B2, B5, B6, B9, B10, B11, and B12 subfamilies, while the R-Ras–binding residues are highly conserved in the B3 (including M68), B4, B7, and B8 subfamilies. Therefore, TcdB seems to have branched into two distinct groups during evolution that have developed two distinct sets of amino acids in their GTPase-binding areas to selectively recognize Rho or R-Ras. We propose to name them the RhoA group and R-Ras group, respectively, based on their preferred substrates, which cause two distinct types of cytopathic effects as reported in prior studies. We envision that these signature sequences in the GTD can be used to predict substrate specificity and pathogenicity of new C. difficile clinical strains that will emerge in the future.

The purified WT and mutant GTDs were transfected into HeLa cell via the Lipofectamine CRISPRMAX Cas9 Transfection Reagent from Thermo Fisher Scientific. The transfection was performed in coherence to the protocol provided by the manufacturer. HeLa cells at 5 × 104 per well were seeded into 24-well plates for overnight culture. For each well, we prepared solutions in tube 1 that contained 25 μl of Opti-MEM medium, 1250 ng of GTD, and 2.5 μl of Cas9 Plus Reagent and in tube 2 that contained 25 μl of Opti-MEM medium and 1.5 μl of CRISPRMAX reagent. We immediately added the solution from tube 1 to tube 2 and then mixed well. We incubated the complex for 10 min at room temperature then added all 50 μl of solution to the cells. The phase-contrast images of cells were taken at the indicated time (Olympus IX51; 10× objective). To test the full-length TcdB, HeLa cells were exposed to WT TcdBVPI10463 or TcdBVPI10463-α16/17M68 at indicated concentrations, and cells were imaged after overnight. Round-shaped and normal-shaped cells were counted manually. The percentage of round-shaped cells was analyzed using the OriginPro (OriginLab, v8.5) and Excel (Microsoft, 2007).

Funding: This work was partly supported by NIH grants R01AI125704, R21AI139690, and R21AI123920 to R.J.; R01NS080833 and R01AI132387 to M.D.; and R01AI139087 and R21 CA235533 to R.J. and M.D. M.D. holds the Investigator in the Pathogenesis of Infectious Disease award from the Burroughs Wellcome Fund. NE-CAT at the Advanced Photon Source (APS) is supported by a grant from the National Institute of General Medical Sciences (P30 GM124165), and the Eiger 16M detector on the 24-ID-E beamline is funded by an NIH-ORIP HEI grant (S10OD021527). Use of the APS, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under contract no. DE-AC02-06CH11357.

Data and materials availability: Atomic coordinates and structure factors of the Cdc42T35N–GTDVPI10463 and R-RasT61N–GTDM68 complexes have been deposited in the PDB under accession codes 7S0Y and 7S0Z, respectively. The x-ray diffraction data were processed with XDS as implemented in RAPD (https://github.com/RAPD/RAPD). All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.

34. Chen P., Lam K. H., Liu Z., Mindlin F. A., Chen B., Gutierrez C. B., Huang L., Zhang Y., Hamza T., Feng H., Matsui T., Bowen M. E., Perry K., Jin R.,

Wild type and mutated recombinant full-length activated BoNT/A1 were produced under biosafety level 2 containment (project number GAA A/Z 40654/3/123) recombinantly in K12 E. coli strain in Dr. Rummel’s lab6-tag were purified on Co2+-Talon matrix (Takara Bio Europe S.A.S., France) and eluted with 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 250 mM imidazole. For proteolytic activation, BoNT/A1 was incubated for 16 h at room temperature with 0.01 U bovine thrombin (Sigma-Aldrich Chemie GmbH, Germany) per µg of BoNT/A1. Subsequent gel filtration (Superdex-200 SEC; GE Healthcare, Germany) was performed in phosphate buffered saline (pH 7.4).

The purified ciA-D12 (S124C) was labeled with Alexa Fluor C2 647 maleimide (Molecular Probes) according to the manufacturer’s instructions. The labeled ciA-D12 was further purified by MonoQ ion-exchange chromatography in 10 mM Hepes (pH 8.0) and eluted with a NaCl gradient. The calculated dye to protein ratio was ~1 mole of dye per mole of ciA-D12. BoNT/A1i-ciA-D12 complex were prepared by mixing BoNT/A1i and ciA-D12 in 1:1.5 molar ratio and the complex was purified by size-exclusion chromatography using Superdex-200.

Liposomes containing 10% GT1b, 69% DOPC, 20% DOPS, 0.5% rhodamine-PE, and 0.5% biotin-PE were prepared by extrusion through a 100 nm pore membrane. To form proteoliposomes, 10 nM BoNT/A1i–D12* or oxidized BoNT/A1iDS–D12* was incubated with 0.5 mg/ml lipid at room temperature for 1 h at the pH indicated. The mixture was then diluted 1000-fold and incubated for 5 minutes in a passivated, quartz microscope chamber functionalized with streptavidin. The biotinylated liposomes were retained and unbound proteins are washed away by exhaustive rinsing with buffer. At the low densities needed for optical resolution of individual liposomes, we could observe sufficient liposomes for statistical analysis, while minimizing the probability that a diffraction-limited spot would contain multiple liposomes. Samples were imaged using a prism-based Total Internal Reflection Fluorescence (TIRF) microscope. Samples were first excited with a laser diode at 640 nm (Newport Corporation, Irvine, CA) to photobleach Alexa 647-labeled BoNT/A1i–D12* or BoNT/A1iDS-D12* molecules followed by excitation with a diode pumped solid state laser at 532 nm (Newport Corporation, Irvine, CA) to probe for Rhodamine-labeled liposomes. Emission from protein and lipids was separated using an Optosplit ratiometric image splitter (Cairn Research Ltd., Faversham UK) containing a 645 nm dichroic mirror, a 585/70 band pass filter for Rhodamine, and a 670/30 band pass filter for Alexa 647 (all Chroma, Bellows Falls, VT). The replicate images were relayed to a single iXon EMCCD camera (Andor Technologies, Belfast, UK) at a frame rate of 10 Hz. Data were processed in MATLAB to cross-correlate the replicate images and extract time traces for diffraction-limited spots with intensity above baseline. Single-molecule traces were hand selected based on the exhibition of single-step decays to baseline during 640 illumination and the appearance of Rhodamine emission during 532 illumination.

This new method of drug development relies on the integration of computer modeling and laboratory testing to rapidly generate and evaluate tens of thousands of potential mini-protein binders with varying shapes. This unprecedented scale of de novo protein design was made possible by recent advances in both the Rosetta software suite and DNA manufacturing. Using this method, mini-protein binders can be rapidly programmed to target a range of proteins, including other viruses, toxins, or even tumor-specific markers.

Aaron Chevalier*, Daniel-Adriano Silva*, Gabriel J. Rocklin*, Derrick R. Hicks, Renan Vergara, Patience Murapa, Steffen M. Bernard, Lu Zhang, Kwok-Ho Lam, Guorui Yao, Christopher D. Bahl, Shin-Ichiro Miyashita, Inna Goreshnik, James T. Fuller, Merika T. Koday, Cody M. Jenkins, Tom Colvin, Lauren Carter, Alan Bohn, Cassie M. Bryan, D. Alejandro Fernández-Velasco, Lance Stewart, Min Dong, Xuhui Huang, Rongsheng Jin, Ian A. Wilson, Deborah H. Fuller& David Baker.

This work was partly supported by National Institute of Allergy and Infectious Diseases (NIAID) grants R01AI139087, R01AI158503, and R21AI156092 to RJ. NE-CAT at the Advanced Photon Source (APS) is supported by a grant from the National Institute of General Medical Sciences (P30 GM124165). Use of the APS, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357.

3. CDC. Antibiotic resistance threats in the united states, 2019. U.S. Department of Health and Human Services C (2019). Available at: https://www.cdc.gov/drugresistance/biggest-threats.html.

22. Chen B, Liu Z, Perry K, Jin R. Structure of the glucosyltransferase domain of tcda in complex with rhoa provides insights into substrate recognition. Sci Rep (2022) 12(1):9028. doi: 10.1038/s41598-022-12909-8

32. Kroh HK, Chandrasekaran R, Rosenthal K, Woods R, Jin X, Ohi MD, et al. Use of a neutralizing antibody helps identify structural features critical for binding of clostridium difficile toxin tcda to the host cell surface. J Biol Chem (2017) 292(35):14401–12. doi: 10.1074/jbc.M117.781112

49. Chen P, Jin R. Receptor binding mechanisms of clostridioides difficile toxin b and implications for therapeutics development. FEBS J (2021). doi: 10.1111/febs.16310

51. Kroh HK, Chandrasekaran R, Zhang Z, Rosenthal K, Woods R, Jin X, et al. A neutralizing antibody that blocks delivery of the enzymatic cargo of clostridium difficile toxin tcdb into host cells. J Biol Chem (2018) 293(3):941–52. doi: 10.1074/jbc.M117.813428

59. Lam K-H, Perry K, Shoemaker CB, Jin R. Two vhh antibodies neutralize botulinum neurotoxin E1 by blocking its membrane translocation in host cells. Toxins (Basel) (2020) 12(10):616. doi: 10.3390/toxins12100616

Citation:Lee K, Gu S, Jin L, Le TTN, Cheng LW, Strotmeier J, et al. (2013) Structure of a Bimodular Botulinum Neurotoxin Complex Provides Insights into Its Oral Toxicity. PLoS Pathog 9(10):

Structural information of HAs is available for serotypes C and D, such as the crystal structures of HA33 of serotype C (HA33-C) [12], [13], a complex composed of HA17 and HA33 of serotype D [14], and HA70 of serotype C (HA70-C) [15], [16]. However, BoNT/C and D rarely cause human botulism but are known to cause the syndrome in cattle, poultry, and wild birds. For BoNT/A, the major cause of human botulism, only the structure of HA33 (HA33-A), which displays an amino-acid identity of ∼38% to HA33-C and D, has been solved [17]. We have recently determined the crystal structure of the BoNT/A–NTNHA complex [5]. However, it remains largely unclear how the HAs assemble with one another and how they interact with BoNT and NTNHA.

Interacting residues are labeled in open-book views of the interfaces. (A) Interface between HA70 and HA17. (B) Interfaces between HA17 and the two HA33s are indicated by purple and blue. The HA33 residues involved in both interfaces are in green. (C) Interface between two HA33s attached to the same HA17. See Fig. S5 and S6 in Text S1 for stereo versions of the detailed interactions.

To determine the physiological relevance of these HA–glycan interactions during toxin absorption, we examined their ability to interfere with the HA complex-mediated disruption of Caco-2 TER. Lac, Gal, and IPTG markedly inhibited the TER reduction induced by the HA complex, and showed higher potencies when applied to the apical than to the basolateral compartment (Fig. 5A–B and Fig. S11A–D in Text S1). In contrast, α2,3- and α2,6-SiaLac, and to a lesser extent Neu5Ac, inhibited the decrease in TER only when applied apically, albeit more weakly than Lac (Fig. 5A–B and Fig. S11E–F in Text S1). We then examined the transport of the HA complex across the Caco-2 monolayer using a fluorescence-labeled HA complex (HA*) (Fig. 5C). Lac and IPTG efficiently inhibited the transport of HA* when it was applied to the apical or basolateral chamber. Blocking the transport of HA* via α2,3- and α2,6-SiaLac was more potent toward the basolateral compartment than toward the apical side. Neu5Ac at 50 mM did not inhibit transport of HA* from either side of the Caco-2 cell monolayer. These data are consistent with the ability of these carbohydrates to inhibit TER reduction induced by the HA complex. Collectively, these results suggest that the binding of HAs to Neu5Ac- and Gal-containing glycans on epithelial cells is essential for the transport of BoNT across the intestinal wall. Moreover, the carbohydrate receptors may play a more important role in the initial L-PTC absorption in the intestinal lumen, whereas other host receptors (e.g., E-cadherin) are involved once it gains access to the basolateral side.

(A, B) TER of Caco-2 monolayers was measured when Alexa-488-labeled HA complex (HA*) pre-incubated with Lac, IPTG, α2,3-SiaLac, or α2,6-SiaLac was applied to the apical (A; 58 nM) or basolateral (B; 17 nM) chambers. Values are means ± SD (n = 4–12). (C) HA* (with or without carbohydrates) or the Alexa-488-labeled HA33-DAFA complex (HA33-DAFA *) was applied to the apical (at 58 nM) or basolateral (at 17 nM) chamber. The fluorescence signals in both chambers were quantified after 24 hours and the amount of transported HA*/HA33-DAFA * was expressed as a percentage of the total HA*/HA33-DAFA * used. Values are means ± SD (n = 3–22).

The loose linkage between the M-PTC and the HA complex clearly suggests divided functions. We previously reported that the M-PTC"s compact structure protects BoNT against digestive enzymes and the extreme acidic environment of the GI tract [5], [23]. We now show that the HA complex is mainly responsible for BoNT absorption in the small intestine, through binding to specific host carbohydrate receptors. This new finding permitted the identification of IPTG as a prototypical oral inhibitor that extends survival following lethal oral BoNT/A intoxication of mice. Multivalent interactions involving nine binding sites for Neu5Ac- and Gal-containing glycans increase the overall avidity of binding between the L-PTC and glycans on the epithelial cell surface, and thus compensate for the modest glycan-binding affinities at individual binding sites (Fig. 6C). Similarly, the potency of carbohydrate receptor mimics could be improved by optimizing the HA–glycan interactions as revealed here or by introducing new HA–inhibitor interactions at individual binding sites based on rational design, as well as by designing multivalent inhibitors. Although such inhibitors cannot be used to treat fully developed food-borne botulism, they could provide temporary protection upon pre-treatment and could also be useful for cases of intestinal colonization with C. botulinum spores such as in cases of infant or adult intestinal botulism. Our results also suggest that the L-PTC could be exploited for alternative applications. For example, protein-based therapeutics could be coupled to the modified non-toxic L-PTC to allow oral delivery by improving drug stability, absorption efficiency, and bioavailability.

The purified HA70 was labeled with Alexa Fluor® 488 carboxylic acid, succinimidyl ester (Life Technologies) according to the manufacturer"s instructions. The labeled HA70 was further purified by Superdex 200 chromatography in 20 mM Tris (pH 8.0) and 50 mM NaCl. The calculated dye to protein ratio was ∼2 moles of dye per mole of monomeric HA70.

The HA17–HA33, the HA70–HA17, and the HA70D3–HA17 complexes were produced by co-expression and co-purification as described above. To assemble the mini-HA complex (HA70D3–HA17–HA33), the purified HA33 and the HA70D3–HA17 complex were mixed at a molar ratio of ∼2.5∶1 and incubated at 4°C overnight. The excess HA33 was removed by Superdex 200 chromatography with 20 mM Tris (pH 7.6) and 50 mM NaCl. The fully assembled HA complex was reconstituted by mixing the purified HA70 and the HA17–HA33 complex at a molar ratio of ∼1∶1.3. The mixture was incubated at 4°C overnight and the excess HA17–HA33 complex was removed from the mature HA complex by Superdex 200 chromatography with 20 mM Tris (pH 7.6) and 50 mM NaCl. The fluorescence-labeled HA complex was prepared with Alexa Fluor® 488-labeled HA70 and unlabeled HA17–HA33 complex (HA*) or HA17–HA33DAFA complex (HA33DAFA*) using a similar protocol.

Sedimentation equilibrium (SE) experiments were performed in a ProteomeLab XL-I (BeckmanCoulter) analytical ultracentrifuge. Purified HA samples were dialyzed extensively against a buffer containing 50 mM Tris (pH 7.6) and various NaCl concentrations, or 50 mM citric acid (pH 2.3) and various NaCl concentrations. Protein samples at concentrations of 0.4, 0.2, and 0.1 unit of OD280 were loaded in 6-channel equilibrium cells and centrifuged at 20°C in an An-50 Ti 8-place rotor at the first speed indicated until equilibrium was achieved and thereafter at the second speed. HA33 was analyzed at rotor speeds of 19,000 and 22,000 rpm. The HA17–HA33 and the HA70D3–HA17–HA33 complexes were analyzed at 12,000 and 14,000 rpm. The HA70–HA17 and the HA70–HA17–HA33 complexes were run at speeds of 6,000 and 8,000 rpm. For each sample, data sets for the two different speeds were analyzed independently using HeteroAnalysis software (by J.L. Cole and J.W. Lary, University of Connecticut). Three independent experiments were performed for each sample.

The L-PTC of BoNT/A was obtained from List Biological Laboratories, Inc. (Campbell, California) and Miprolab GmbH (Göttingen, Germany). The recombinant HA complex was reconstituted in vitro as described above. Negatively stained EM specimens were prepared following a previously described protocol [54]. Briefly, 3 µl of the L-PTC (∼0.02 mg/ml in 20 mM MES, pH 6.2, and 100 mM NaCl) or the HA complex (∼0.01 mg/ml in 20 mM Tris, pH 7.6, and 50 mM NaCl) was placed on a freshly glow-discharged carbon-coated EM grid, blotted with filter paper after 40 seconds, washed with two drops of deionized water, and then stained with two drops of freshly prepared 1% uranyl formate, which also served to fix the proteins.

Carbohydrate inhibition assays: Lac, Gal, IPTG, Neu5Ac, α2,6- and α2,3-SiaLac were dissolved in IMDM, sterile filtered and stored at −20°C. Neu5Ac stock solution was adjusted to pH 7.4. The wild type HA complex (HA wt), fluorescence-labeled HA complex (HA*), or the HA70-TPRA complex were pre-incubated with the corresponding carbohydrate over night at 4°C in IMDM and diluted to the final concentration with IMDM prior to administration. The TER upon administration of each carbohydrate in the highest concentrations used was checked in the absence of HA and was virtually identical to that of the control without sugars.

Transport measurement: For paracellular transport studies, filters were incubated in IMDM added to the apical (0.5 ml) and basolateral (1.5 ml) reservoir. As marker substance Alexa Fluor® 488 labeled HA* or HA33-DAFA* was administered to the apical or basolateral reservoirs at final concentrations of 58 nM and 17 nM, respectively. After 24 hour of incubation, 200 µl of samples were taken from the apical and the basolateral reservoir. The marker substance was measured in a BioTek Synergy 4 fluorescence spectrophotometer at 495 nm excitation and 519 nm emission wavelengths.

4.Cheng LW, Onisko B, Johnson EA, Reader JR, Griffey SM, et al. (2008) Effects of purification on the bioavailability of botulinum neurotoxin type A. Toxicology 249: 123–129.

20.Inoue K, Fujinaga Y, Watanabe T, Ohyama T, Takeshi K, et al. (1996) Molecular composition of Clostridium botulinum type A progenitor toxins. Infect Immun 64: 1589–1594.

25.Fujinaga Y, Inoue K, Nomura T, Sasaki J, Marvaud JC, et al. (2000) Identification and characterization of functional subunits of Clostridium botulinum type A progenitor toxin involved in binding to intestinal microvilli and erythrocytes. FEBS Lett 467: 179–183.

26.Inoue K, Fujinaga Y, Honke K, Arimitsu H, Mahmut N, et al. (2001) Clostridium botulinum type A haemagglutinin-positive progenitor toxin (HA(+)-PTX) binds to oligosaccharides containing Gal beta1-4GlcNAc through one subcomponent of haemagglutinin (HA1). Microbiology 147: 811–819.

27.Sugawara Y, Matsumura T, Takegahara Y, Jin Y, Tsukasaki Y, et al. (2010) Botulinum hemagglutinin disrupts the intercellular epithelial barrier by directly binding E-cadherin. J Cell Biol 189: 691–700.

28.Jin Y, Takegahara Y, Sugawara Y, Matsumura T, Fujinaga Y (2009) Disruption of the epithelial barrier by botulinum haemagglutinin (HA) proteins - differences in cell tropism and the mechanism of action between HA proteins of types A or B, and HA proteins of type C. Microbiology 155: 35–45.

29.Matsumura T, Jin Y, Kabumoto Y, Takegahara Y, Oguma K, et al. (2008) The HA proteins of botulinum toxin disrupt intestinal epithelial intercellular junctions to increase toxin absorption. Cell Microbiol 10: 355–364.

32.Fujita R, Fujinaga Y, Inoue K, Nakajima H, Kumon H, et al. (1995) Molecular characterization of two forms of nontoxic-nonhemagglutinin components of Clostridium botulinum type A progenitor toxins. FEBS Lett 376: 41–44.

33.Ohyama T, Watanabe T, Fujinaga Y, Inoue K, Sunagawa H, et al. (1995) Characterization of nontoxic-nonhemagglutinin component of the two types of progenitor toxin (M and L) produced by Clostridium botulinum type D CB-16. Microbiol Immunol 39: 457–465.

This work was partly supported by National Institute of Allergy and Infectious Diseases (NIAID) grants R01AI139087, R01AI158503, and R21AI156092 to RJ. NE-CAT at the Advanced Photon Source (APS) is supported by a grant from the National Institute of General Medical Sciences (P30 GM124165). Use of the APS, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02-06CH11357.

3. CDC. Antibiotic resistance threats in the united states, 2019. U.S. Department of Health and Human Services C (2019). Available at: https://www.cdc.gov/drugresistance/biggest-threats.html.

22. Chen B, Liu Z, Perry K, Jin R. Structure of the glucosyltransferase domain of tcda in complex with rhoa provides insights into substrate recognition. Sci Rep (2022) 12(1):9028. doi: 10.1038/s41598-022-12909-8

32. Kroh HK, Chandrasekaran R, Rosenthal K, Woods R, Jin X, Ohi MD, et al. Use of a neutralizing antibody helps identify structural features critical for binding of clostridium difficile toxin tcda to the host cell surface. J Biol Chem (2017) 292(35):14401–12. doi: 10.1074/jbc.M117.781112

49. Chen P, Jin R. Receptor binding mechanisms of clostridioides difficile toxin b and implications for therapeutics development. FEBS J (2021). doi: 10.1111/febs.16310

51. Kroh HK, Chandrasekaran R, Zhang Z, Rosenthal K, Woods R, Jin X, et al. A neutralizing antibody that blocks delivery of the enzymatic cargo of clostridium difficile toxin tcdb into host cells. J Biol Chem (2018) 293(3):941–52. doi: 10.1074/jbc.M117.813428

59. Lam K-H, Perry K, Shoemaker CB, Jin R. Two vhh antibodies neutralize botulinum neurotoxin E1 by blocking its membrane translocation in host cells. Toxins (Basel) (2020) 12(10):616. doi: 10.3390/toxins12100616

Figure 2. Intestinal binding of the PTC is primarily mediated by the HA complex. Same amount of the myc-tagged HA-wt, HA-mini, HA-DAFA, M-PTC, and non-toxic non-hemagglutinin (NTNHA) were incubated with mouse jejunum segments separately. The tissues were fixed, immunostained with a monoclonal anti-myc antibody, and visualized with a secondary antibody conjugated with Alexa Fluor 594 (red). The nuclei of the epithelial cells were labeled with DAPI (blue). The HA-wt complex showed a robust binding on luminal surface of the small intestine at both pH (A) 6.5 and (C) 7.4. In contrast, the (B) M-PTC and (D) NTNHA did not have detectable binding signal. (E) The HA-mini complex showed a similar binding pattern and intensity in comparison to the HA-wt complex. (F) The HA-DAFA mutant showed a significantly decreased binding. Images with 3x magnification were shown to the right of each panel. A minimum of three slides were prepared and analyzed for each sample, and the experiments were repeated using two mice. One slide of each sample was shown as representative. Scale bars: 0.2 mm.

In spite of a wealth of information on HA-carbohydrate interactions [22-26], the receptor-binding specificity of the HA proteins has not been systematically addressed. To fill in the knowledge gap, we performed comprehensive glycan array screenings at the Core H of the Consortium for Functional Glycomics (CFG) using the mammalian glycan array (version 5.1) that contains a broad-spectrum of natural/synthetic glycans (610 glycans in total). An Alexa Fluor® 488-labeled HA70 was analyzed at 1000 and 200 ^g/mL concentrations (equivalent to 4.8 and 0.96 ^M). The complete HA-wt complex containing the labeled HA70 was probed at concentrations of 1000, 100, 30 and 10 p,g/mL (equivalent

The fixed tissues were embedded in optimum cutting temperature (O.C.T.) compound in a cryo-mold placed in a 2-methylbutane bath that was precooled by dry ice. After embedding, the samples were stored at -70 °C. Before section, the tissues were placed in the cryostat for 1 h to warm up to -20 °C. Frozen sections were rinsed three times with PBS (pH 7.4), and incubated with primary antibody (anti-myc tag antibody, Abcam, Cambridge, UK) at room temperature for 1 h. After washing with three changes of PBS, the secondary antibody (Alexa Fluor anti Rabbit IgG 594, Invitrogen, Carlsbad, CA, USA) was applied and incubated with the sections for 30 min at room temperature. Samples were washed three times with PBS and then coverslipped with VectaShield Hardset Mounting Medium with DAPI (Vector Labs, Burlingame, CA, USA). All slides were scanned at a magnification of 20* using the Aperio Scanscope FL system (Aperio Technologies, Vista, CA, USA). The appropriate dyes were assigned and illumination levels were calibrated using a preset procedure, the parameters were saved and applied to all slides.

The purified HA70 was labeled with Alexa Fluor® 488 carboxylic acid, succinimidyl ester (Life Technologies, Carlsbad, CA, USA) according to the manufacturer"s instruction. The labeled HA complex was prepared using the labeled HA70 and the unlabeled HA17-HA33 complex. Before screening, the fluorescently labeled samples were diluted to various concentrations in standard binding buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM CaCh, 2 mM MgCh, 1% BSA, and 0.05% Tween 20). The screening was performed at the Core H of the CFG using the mammalian glycan array (version 5.1) [28].

We thank Robbin Newlin for assistance with tissue preparation and immunostaining. We thank Core H of the Consortium for Functional Glycomics (CFG) for glycan array screening. This work was partly supported by National Institute of Allergy and Infectious Diseases (NIAID) grant R01AI091823 to Rongsheng Jin.

13.Cheng, L.W.; Onisko, B.; Johnson, E.A.; Reader, J.R.; Griffey, S.M.; Larson, A.E.; Tepp, W.H.; Stanker, L.H.; Brandon, D.L.; Carter, J.M. Effects of purification on the bioavailability of botulinum neurotoxin type A. Toxicology 2008, 249, 123-129.

16.Gu, S.; Rumpel, S.; Zhou, J.; Strotmeier, J.; Bigalke, H.; Perry, K.; Shoemaker, C.B.; Rummel, A.; Jin, R. Botulinum neurotoxin is shielded by NTNHA in an interlocked complex. Science 2012, 335, 977-981.

17.Lee, K.; Gu, S.; Jin, L.; Le, T.T.; Cheng, L.W.; Strotmeier, J.; Kruel, A.M.; Yao, G.; Perry, K.; et al. Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity. PLoSPathog. 2013, 9, e1003690.

18.Amatsu, S.; Sugawara, Y.; Matsumura, T.; Kitadokoro, K.; Fujinaga, Y. Crystal structure of Clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular complex. J. Biol. Chem. 2013, 288, 35617-35625.

22.Fujinaga, Y.; Inoue, K.; Watanabe, S.; Yokota, K.; Hirai, Y.; Nagamachi, E.; Oguma, K. The haemagglutinin of Clostridium botulinum type C progenitor toxin plays an essential role in binding of toxin to the epithelial cells of guinea pig small intestine, leading to the efficient absorption of the toxin. Microbiology 1997, 143, 3841-3847.

23.Nishikawa, A.; Uotsu, N.; Arimitsu, H.; Lee, J.C.; Miura, Y.; Fujinaga, Y.; Nakada, H.; Watanabe, T.; Ohyama, T.; Sakano, Y.; et al. The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells. Biochem. Biophys. Res. Commun. 2004, 319, 327-333.

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the most poisonous biological toxins for humans, which potently block neurotransmitter release (Rossetto, Pirazzini and Montecucco 2014). They adopt a similar architecture composed of a ∼50 kDa light chain (LC) and a ∼100 kDa C-terminal heavy chain (HC), which are linked by a disulfide bridge. HC could be further divided into a translocation domain (HN) that delivers LC to neuronal cytosol, and a receptor-binding domain (HC) that specifically recognizes motoneurons. LC is a Zn2+-containing protease that cleaves unique scissile peptide bonds in soluble N-ethylmaleimide-sensitive fusion protein attachment receptor (SNAREs) proteins, including synaptosome associated protein of 25 kDa (SNAP-25), vesicle associated membrane protein (VAMP) (also called synaptobrevin) and syntaxin. Cleavage of SNARE proteins inhibits the release of neurotransmitter acetylcholine and subsequently paralyzes the affected muscles (Turton, Chaddock and Acharya 2002; Brunger, Jin and Breidenbach 2008; Rossetto et al.2013; Pantano and Montecucco 2014).

Cleavage assays were performed as previously described (Sikorra et al.2008). Briefly, rat VAMP-2 and its mutants were generated by in vitro transcription/translation. Cleavage assays contained 1 μl of the transcription/translation mixture of [35S]methionine-labeled wild-type or mutated VAMP-2 and purified LC (LC/F5 and LC/HA at 100 and 50 nM final concentrations, respectively) and were incubated for 60 min at 37°C in a total volume of 10 μl of toxin assay buffer.

For determination of the enzyme kinetic parameters, VAMP-2 concentration was varied between 3 and 130 μM using E. coli expressed His6-VAMP-2. Each of the various substrate concentrations was endowed by the addition of 1 μl of radiolabeled His6-VAMP-2 generated by in vitro transcription/translation. LC/F5 and LC/HA were used at final concentrations of 2 and 10 nM, respectively. Incubation was done in a final volume of 25 μl of toxin assay buffer. After 2 and 4 min of incubation at 37°C, aliquots of 10 μl were taken.

Enzymatic reactions were stopped by mixing with 10 μl of pre-chilled double-concentrated SDS-PAGE sample buffer. VAMP-2 and its cleavage products were separated by SDS-PAGE, and radiolabeled protein was visualized using a FLA-9000 image scanner (Fuji Photo Film, Co., Ltd., Tokyo, Japan). The percentage of hydrolyzed VAMP-2 was determined from the turnover of the radiolabeled substrate applying the Multigauge 3.2 software (Fuji Photo Film) and used to calculate the initial velocity of substrate hydrolysis. KM and Vmax values were calculated by non-linear regression using the GraphPad Prism 4.03 program (GraphPad Software Inc., San Diego, CA).

Overall structure of LC/HA. (A) Cartoon representation of an LC/HA crystallographic dimer. The Zn2+- and Ca2+-coordinating residues are drawn as sticks and labeled. Ca2+ and Zn2+ are shown as silver and green spheres, respectively. (B) Structural superposition of LC/HA and the LCs of seven established BoNT serotypes and TeNT: LC/A (PDB code: 1XTF) (Breidenbach and Brunger 2004), white; LC/B (2ETF), salmon; LC/C (2QN0) (Jin et al.2007), slate; LC/D (2FPQ) (Arndt et al.2006), orange; LC/E (1T3A) (Agarwal et al.2004), yellow; LC/F1 (2A97) (Agarwal, Binz and Swaminathan 2005), magenta; LC/G (1ZB7) (Arndt et al.2005), lime; TeNT-LC (1YVG) (Rao et al.2005), forest, LC/HA, cyan. (C) The active site of LC/HA. Two acetate ions identified near the active site are drawn as pink sticks and two interacting water molecules are shown as red spheres.

The VAMP-2 variants were produced as radiolabeled molecules by in vitro transcription/translation and incubated for 1 h with LC/HA or LC/F5. The percentage of cleavage of the VAMP-2 mutants versus that of the wild-type VAMP-2 was calculated. The amino acid substitutions that resulted in at least 33% diminished cleavage rate were considered critical for substrate cleavage. We observed significant effects when VAMP-2 residues between S28 and K59 were mutated, which include residues around the SNARE secondary recognition motif (SSR motif) V1 (residues 39–47), but not the V2 (residues 62–71) (Rossetto et al.1994). This finding suggests that LC/HA and LC/F5 require a substrate segment spanning 32 amino acids surrounding the cleavage site for optimal cleavage.

Cleavage analysis of various VAMP-2 point mutants. (A) Schematic representation of VAMP-2 and the mutations introduced in a segment between residues T27 to D68. The scissile peptide bonds are marked for LC/HA and LC/F1. (B, C) Cleavage assays. His6-VAMP-2 (1–96) variants were radiolabeled by in vitro transcription/translation and incubated for 1 h in the presence of 50 nM LC/HA, 100 nM LC/F5. Samples were analyzed by Tris/Tricine-PAGE using 15% gels. Columns represent percentages of cleavage caused by the mutants versus the wild-type VAMP-2. Data represent means ± SD of at least four independent experiments. The color code applied to the columns is as follows: green, no or less than 10% reduction of cleavability; yellow, >10%; pink, >33%; red, >66%. A peptide segment between R47 and K59 that may adopt an extended conformation is highlighted with a red line.

Since the crystal structure of LC/F5 is not available, we generated a model of LC/F5 using LC/HA as a template for further structural analysis. As expected, the overall electrostatic features of these two LCs around the active site are very similar (Fig. 3A). However, they exhibit distinct surface charge distribution in a potential VAMP-2 exosite-binding pocket, which is predicted based on the structure of the LC/F1-VAMP-2 complex (Agarwal et al.2009). This area bears more negative charges in LC/HA, but more positive charge in LC/F5 (Fig. 3B). We speculate that this difference may contribute to the different affinity of LC/HA and LC/F5 towards VAMP-2.

Remarkably, we found that mutating the P1 residue L54 to Ala enhanced the VAMP-2 cleavability by ∼2.5 fold in LC/F5, but had no effect on LC/HA. The increased VAMP-2 cleavage by LC/F5 is unexpected because earlier studies show that mutations at P1 position usually do not significantly change the VAMP-2 cleavability of other BoNT LCs (Vaidyanathan et al.1999; Jin et al.2007; Sikorra et al.2008). This finding thus suggests that the P1 residue L54 probably is not directly involved in positioning the scissile peptide bond in the active sites of LC/F5, and it might even impose a steric hindrance for VAMP-2 binding to LC/F5.

The overall structures of LC/HA and LC/F1 are very similar, but LC/HA adopts different conformations in several surface loops, including the 60, 170, 210 and 250 loops and the C-terminus (Fig. 4A). The conformations of the 60, 170 and 250 loops in LC/HA may be affected by crystal packing because they interact with a neighboring symmetric LC/HA molecule, but the 210 loop and the C-terminal helix of LC/HA do not. In LC/HA, the 60 loop, 210 loop and the C-terminal helix are more structured, while the 170 loop and 250 loop show higher structure flexibility. In particular, the 250 loop of LC/HA is largely unstructured while the corresponding loop forms an anti-parallel β-hairpin in LC/F1, LC/A and LC/C (Breidenbach and Brunger 2004; Jin et al.2007; Agarwal et al.2009). It is known that the 60 and 250 loops in LC/A1 bind SNAP-25 at a site C-terminal to the cleavage site, and the 170 loop in LC/F1 binds VAMP-2 at a site N-terminal to the cleavage site (Agarwal et al.2009). Therefore, these unique conformations observed in LC/HA may indicate different VAMP-2 binding modes between LC/HA and LC/F1, which may partly contribute to their preference for different VAMP-2 cleavage sites.

Structural comparison between LC/HA and LC/F1. (A) Structural superposition between LC/HA and LC/F1, and areas that show different conformations are colored in orange for LC/HA and blue for LC/F1. (B) A structure model of the LC/HA-VAMP-2 (R47 – K59) complex. Atoms are colored to highlight hydrophobicity features (Hagemans et al.2015): carbon atoms not bound to oxygen or nitrogen atoms are colored orange, nitrogen atoms carrying positive charges in arginine and lysine are blue, oxygen atoms carrying negative charges in glutamate and aspartate are red, and all remaining atoms are white. A structural model of VAMP-2 is colored green. (C) Close-up view of the LC/HA-VAMP-2 binding surface. Key residues of LC/HA predicted to interact with VAMP-2 are labeled. (D) The structure of LC/F1-VAMP-2 complex (PDB code: 3FIE). The color scheme for LC/F1 is the same as that shown in panel (B), and the VAMP-2 peptide is shown as a wheat cartoon loop. Black arrows mark an electropositive VAMP-2-binding pocket in LC/F1, whereas the corresponding surface in LC/HA displayed different charge property. (E) Close-up view of the LC/F1-VAMP-2 binding surface. Residues that contribute to the unique electropositive surface patch in LC/F1 are labeled.

In summary, we present here the first comprehensive structural and biochemical analysis of the substrate-binding mechanism of LC/HA. Our findings demonstrate that LC/HA recognizes VAMP-2 in a way that is distinct from other VAMP-2-specific BoNTs. Most notably, VAMP-2 may adopt an extended conformation in a segment surrounding the scissile peptide bond, which involves more side-chain mediated interactions with the active site of LC/HA to ensure a proper positioning of the scissile peptide bond. Furthermore, the different surface electrostatic properties of LC/HA and LC/F1 in the VAMP-2 binding groove might account for their different cleavage sites. Our structural model could facilitate the development of peptide or small molecule inhibitors targeting LC/HA. Such inhibitors against the LC of BoNT will be important for clinically relevant post-intoxication treatment, which will greatly complement the currently available treatments that use neutralizing antibodies to clear the toxins in the blood stream.

This work was partly supported by National Institute of Health (NIH) grants R01AI091823, R01 AI125704 and R21AI123920 to RJ and by grant BI 660/3–1 from the Deutsche Forschungsgemeinschaft to TB. NE-CAT at the Advanced Photon Source (APS) is supported by a grant from the National Institute of General Medical Sciences (P41 GM103403). Use of the APS, an Office of Science User Facility operated for the U.S. Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the U.S. DOE under Contract No. DE-AC02–06CH11357. Coordinates and structure factors of LC/HA have been deposited in the Protein Data Bank under accession code 6BVD.