jin rongsheng uc irvine quotation

Structure and function of bacterial toxins and receptors; synaptic proteins; protein complexes; protein-protein and protein-ligand interactions; X-ray crystallography; high-throughput screening

Our research group is dedicated to understand the molecular basis of human diseases using structural biology, which allows us to visualize how proteins function or malfunction at the atomic level. Our current research is focused on three areas: (1) exploring the molecular mechanisms underlying the toxic and therapeutic functions of botulinum neurotoxins (BoNTs), which will help to develop effective anti-BoNT strategies and improve clinical applications of BoNT; (2) characterizing the structures of Clostridium difficile (C. diff) toxins (TcdA and TcdB) and their interactions with host receptors, and understanding how they contribute to the disease of Clostridium difficile infection (CDI) that tops the CDC’s list of urgent threats; (3) advancing mechanistic understanding of ion channels, receptors, and signaling molecules in the nervous system, which will facilitate the design and improvement of therapeutic agents for the treatment of some psychological and neurological disorders. We are also developing cutting-edge small molecule high-throughput screening (HTS) assays based on our understanding of the structure and function of these disease-related proteins, which may lead to novel chemical probes and/or drug candidates for basic research and therapeutic application.

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)

Structure of the glucosyltransferase domain of TcdA in complex with RhoA provides insights into substrate recognition. Sci Rep. 2022 05 30; 12(1):9028.

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.

Probing the structure and function of the protease domain of botulinum neurotoxins using single-domain antibodies. PLoS Pathog. 2022 01; 18(1):e1010169.

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.

Structural Insights into Rational Design of Single-Domain Antibody-Based Antitoxins against Botulinum Neurotoxins. Cell Rep. 2020 02 25; 30(8):2526-2539.e6.

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.

The hypothetical protein P47 of Clostridium botulinum E1 strain Beluga has a structural topology similar to bactericidal/permeability-increasing protein. Toxicon. 2018 Jun 01; 147:19-26.

High-resolution crystal structure of HA33 of botulinum neurotoxin type B progenitor toxin complex. Biochem Biophys Res Commun. 2014 Apr 04; 446(2):568-73.

Join us for#STATMadnessChatstomorrow at 1 p.m. PT to learn more about Dr. Rongsheng Jin’s work, “attacking C. diff toxin.” And, don’t forget to keep casting your votes for UCI in#STATMadnessathttps://bit.ly/2WzFYkZ!@UofCAHealth@UCIrvine@UCIrvineHealth@UofCalifornia

Botulinum neurotoxins (BoNTs) are extremely poisonous protein toxins that cause the fatal paralytic disease botulism. They are naturally produced in bacteria with several nontoxic neurotoxin-associated proteins (NAPs) and together they form a progenitor toxin complex (PTC), the largest bacterial toxin complex known. In foodborne botulism, the PTC functions as a molecular machine that helps BoNT breach the host defense in the gut. Here, we discuss the substantial recent advance in elucidating the atomic structures and assembly of the 14-subunit PTC, including structures of BoNT and four NAPs. These structural studies shed light on the molecular mechanisms by which BoNT is protected against the acidic environment and proteolytic destruction in the gastrointestinal tract, and how it is delivered across the intestinal epithelial barrier.

By contrast to the well-studied BoNT–neuron interaction, it is not known how BoNTs in foodborne botulism manage to achieve efficient absorption through the gastrointestinal (GI) tract, which is possibly the most challenging route of entry into the systemic circulation. After ingestion with toxin-contaminated food, BoNTs have to tolerate the extremely acidic (pH < 3) and protease-rich environment of the stomach, and the tightly regulated intestinal barrier. We now know that BoNTs overcome the host defense in the form of a large multi-protein complex, the progenitor toxin complex (PTC). The PTC of some BoNT serotypes exhibits ~360–16,000-fold greater oral toxicity than the free BoNT [10-13]. In this review, we summarize recent progress in understanding the structure and assembly of the PTC, emphasizing the structural determinants that guard the toxin when circumventing the primary host defense in the gut.

BoNTs are naturally produced as PTCs, which are composed of BoNT and several auxiliary components termed nontoxic neurotoxin-associated proteins (NAPs). The NAPs are encoded together with the bont gene in one of two different gene clusters, the HA cluster or the orfX cluster [14]. Both clusters encode the non-toxic non-hemagglutinin (NTNHA) protein (Figure 1B), which assembles with BoNT to form the minimally functional PTC (M-PTC, also termed the 12S toxin) (Figure 1C). The HA gene cluster, as observed in BoNT/A–D and G, encodes three hemagglutinins (HA70, HA17, and HA33; also called HA3, HA2, and HA1, respectively) (Figure 1D), which together with BoNT and NTNHA constitute the large PTC (L-PTC or the 16S toxin) (Figure 1E) [15]. By contrast, some BoNTs, such as BoNT/A2–4, E, and F, are encoded in the orfX gene cluster, which contains several orfX genes but not the HA genes. The function of the corresponding orfX proteins remains elusive.

Atomic models of the L-PTC of BoNT/A (L-PTC/A) and BoNT/B (L-PTC/B) have been recently elucidated using a combination of X-crystallography and electron microscopy (EM) [16••,17••]. They display a similar structural organization, which is composed of 14 protein subunits including BoNT, NTNHA, HA70, HA17 and HA33 in a 1:1:3:3:6 stoichiometry. The overall architecture of the L-PTC consists of two structurally and functionally independent entities, an ovoid-shaped M-PTC and a triskelion-shaped HA complex (Figure 1E). The M-PTC protects BoNT from destruction in the GI tract and the HA complex allows BoNT to dock onto the receptors located on the lumen of the small intestine. Based on an earlier EM study, the L-PTC of BoNT/D (L-PTC/D) likely adopts a similar structure [18], suggesting that the L-PTC structure may be conserved across HA-containing BoNT serotypes.

In the absence of M-PTC formation, free BoNT/A is easily inactivated by digestive proteases or by incubation under an acidic environment. Its oral median lethal dose (LD50) is reduced 10-20-fold when it forms the M-PTC with NTNHA. The crystal structure of the M-PTC of BoNT/A offers the first molecular insight into the protection mechanism (Figure 1C) [19••,20]. NTNHA-A has a strikingly similar tripartite architecture as BoNT/A, despite their low amino acid sequence identity. The three domains of NTNHA-A are therefore named as nLC, nHN, and nHC, because they resemble LC, HN, and HC of BoNT/A, respectively. However, BoNT/A residues that are important for its Zn2+-dependent endopeptidase activity and receptor binding are lost in NTNHA-A, which therefore lacks the neurotoxicity. BoNT/A and NTNHA-A form an inter-locked complex that buries a large solvent-accessible area of ~3200 Å per subunit. Interestingly, all three domains of NTNHA-A bind to the HC fragment of BoNT/A, leaving LC largely exposed (Figure 1C), which is consistent with the biochemical finding that HC is more susceptible to proteolytic cleavage than LC and HN [21,22]. Mechanistically, HC-mediated receptor binding is the earliest step during neuron invasion and likely one of the most crucial, because damage to HC would otherwise jeopardize the enrichment of BoNT/A on the neuron membrane [23]. Therefore, the apparently biased molecular safeguard for HC, as opposed to the other toxin domains, is likely the most efficient strategy to protect such a large protein [19••].

Interestingly, BoNT is released from the PTC upon transition from acidic to neutral pH, as occurs during absorption from the intestine into the general circulation [24]. This is achieved by the presence of pH-dependent interactions between BoNT and NTNHA [19••]. Recent small-angle X-ray scattering studies indicate that NTNHA-A is able to sense the change of environmental pH, and that acidic conditions induce NTNHA-A to adopt a specific conformation that initiates a mutual induced fit between NTNHA-A and BoNT/A [25•,26]. At the same time, pH-sensing residues on BoNT/A (e.g., Glu982 and Asp1037) and NTNHA-A are protonated to allow favorable local electrostatic interactions between them to strengthen the binding (Figure 1C) [19••]. The inherent pH sensing feature of the M-PTC is crucial to ensure stable binding to protect BoNT in the GI tract and release it in systemic circulation.

Currently, a high-resolution structure of an M-PTC is only available for BoNT/A. The structures of the M-PTC of BoNT/B and BoNT/E revealed by negative stained EM and 3D reconstruction closely resemble that of BoNT/A [17••]. The crystal structure of the free-form of NTNHA-D is highly similar to NTNHA-A [26]. Therefore, the BoNT–NTNHA binding module is likely conserved across different BoNTs serotypes. It is worth noting that a unique peptide fragment in nLC of NTNHA, termed the nLoop, is conserved in HA-containing BoNT serotypes, and likely mediates the interaction between the M-PTC and the HA complex [19••,27]. But the molecular details of this interaction have yet to be determined.

Atomic models of the fully assembled HA complexes of BoNT/A (HA/A) and BoNT/B (HA/B) have been determined recently [16••,17••,28•]. In addition, the subcomponent structures of HAs are available for BoNT/C (HA33 and HA70) [29-32] and BoNT/D (HA17–HA33 complex) [18]. The HA complex features three prominent triangular blades (Figure 2). The center of the complex is the trimeric HA70 hub. Each HA70 contains three domains (named D1–3): D1 and D2 participate in homo-trimerization and D3 sits at the periphery of the trimer and interacts with HA17. HA17 contains a compact β-trefoil fold and simultaneously binds two HA33 molecules. Each HA33 is composed of two β-trefoil domains linked by an α helix (Figure 2). Although the N-terminal domain of HA33 is restrained by docking to HA17, its C-terminal domain is exposed and exhibits significant conformational plasticity [16••,28•]. The overall structure of the HA complex is likely to be flexible and its three arms may adopt different conformations [17••].

The triskelion-shaped HA complex in complex with carbohydrates and E-cadherin. As highlighted in the right box, each arm of the HA complex contains one HA70 (sand), one HA17 (pink), and two HA33 (orange). HA70 D1 (light blue) and D2 (brown) domains are crucial for the trimer formation of HA70. HA/A contains nine glycan-binding sites with three Neu5Ac-binding sites on HA70 (lime) and six galactose-binding sites on HA33 (forest) (PDB: 4LO1 and 4LO5). HA/C may contain one additional Neu5Ac-binding site on each HA33 (circled). E-cadherin has five extracellular domains (EC1–EC5). To form a trans dimer, residue Trp2 (green sphere) of an E-cadherin (blue molecule) needs to dock into the complementary Trp-binding pocket on the partner E-cadherin (pink molecule). As shown in the left box, the HA complex (surface representation) sequesters E-cadherin (blue molecule) in the monomeric state with the Trp2 (blue sphere) resting in its own Trp-binding pocket, and also blocks the access of its potential dimer partner (PDB: 4QD2).

The intestinal microvilli are covered by a stratified layer of mucus. The HA complex is believed to anchor the L-PTC on the intestinal surface through its multiple carbohydrate-binding sites. HA70 binds one Neu5Ac-containing carbohydrate [16••]. HA33, on the other hand, binds one galactose-containing carbohydrate through its C-terminal β-trefoil domain in serotypes A and B [16••,33•]. HA33 serotype C, however, displays a lower affinity for galactose, but carries an extra Neu5Ac-binding site near the Gal-binding pocket [30,34]. These serotype-specific HA–glycan interactions may partially contribute to the different oral toxicity and host susceptibility among different BoNT serotypes [35-38]. Altogether, each HA complex likely displays multivalent carbohydrate binding, involving up to 9 carbohydrates in L-PTC/A and B and up to 15 carbohydrates in L-PTC/C (Figure 2) [16••,30,34]. Moreover, the carbohydrate-binding domain of HA33 is located at the very tip of the HA complex and displays significant conformational flexibility, which may allow the complex to adjust itself on the intestinal surface to achieve optimal multivalent glycan binding [35,39]. The physiological importance of the HA– carbohydrate interactions has been further validated by in vivo studies using a mouse oral toxicity assay. Carbohydrate receptor mimics (for instance, IPTG) extended survival of mice following lethal BoNT/A oral intoxication [16••], and a mutated L-PTC/A that is unable to bind carbohydrate displayed drastically reduced oral toxicity [40••].

A major advance was provided by the crystal structure of an HA/A subcomplex bound with EC1–EC2 of E-cadherin [40••]. This structure showed that the HA complex stabilizes the A*/A strand of E-cadherin in its monomeric conformation with Trp2 binding intra-molecularly into its own Trp-binding pocket, therefore relieving the driving force for trans-dimerization (Figure 2, left panel). Furthermore, the HA complex occupies the E-cadherin dimer interface that is required to form the trans-dimer and the X-dimer. Consistent with this finding, HA/A binds the monomeric EC1–EC2 domains with an affinity that is much stronger than the affinity of E-cadherin homo-dimerization [40••,44,47]. The model that disruption of the adherens junctions of epithelial cells by the HA complex opens up a paracellular route to facilitate BoNT absorption has been supported by extensive in vitro and ex vivo studies [33•,40••,42] (Figure 3), and was further confirmed by an in vivo study showing that an E-cadherin binding deficient L-PTC/A has markedly decreased oral toxicity in mouse [40••].

Interestingly, the complete triskelion-shape of the HA complex is crucial for its function, because a sub-complex representing one arm of the HA complex failed to disrupt cell-cell junctions [40••]. The fully assembled HA complex might simultaneously bind three E-cadherins, which would greatly strengthen binding through avidity effects and is likely necessary to achieve potent binding in vivo. Furthermore, the bulky triskelion-shaped HA complex (~260 Å wide and ~100 Å height) might disrupt the condensed arrays of E-cadherin dimers that normally stabilize adherens junctions. Additionally, sequestration of E-cadherin by the HA complex might destabilize adherens junctions by affecting the turnover of E-cadherin at adherens junctions [48].

Many structural and functional studies suggest that BoNTs have two different routes of passing through the intestinal epithelial cells (Figure 3). In one scenario, BoNTs in the forms of L-PTC, M-PTC or the free form may cross the epithelial cells by transcytosis without interfering with the epithelial barrier. Once the HA complexes gain access to the basolateral surface, they disrupt E-cadherin-mediated cell-cell adhesion, thereby opening up the paracellular route for BoNT absorption. However, many fundamental questions remain unanswered. For example, the mechanism of BoNT transcytosis is largely unknown. Some data suggest that BoNT might directly recognize specific receptors on intestinal cell surface that mediate transcytosis [51,52]. Alternatively, it is possible that there are transcytosis hot spots on intestinal epithelia, for instance microfold cells (M cells) and neuroendocrine crypt cells, which could mediate BoNT transcytosis [42,53]. Notably, E-cadherin is luminally accessible around mucus-expelling goblet cells, around extruding enterocytes at the tip and lateral sides of villi, and in villus epithelial folds [54]. Hence, the HA complex might access E-cadherin in the intestinal lumen to mediate transcytosis and/or paracellular crossing.

Structures of the 760 kDa L-PTC have revealed a sophisticated macromolecular machine of bacterial toxins that evades host defense systems. Besides stabilizing BoNT in the harsh environment of the GI tract, the L-PTC efficiently delivers BoNT into the general circulation through up to 15 binding sites for cell surface carbohydrates and 3 binding sites for the crucial host adhesion protein E-cadherin. It is worth noting that BoNTs use a dual-receptor mechanism to recognize nerve terminals by interacting with both a protein receptor and gangliosides to mediate cell entry at neuromuscular junctions [23]. It is remarkable that BoNTs use the “same” strategy twice, targeting different host receptors at different times and locations, to ensure its extreme toxicity.

The advances in understanding the structure and function of the L-PTC will promote the development of novel chemical inhibitors or antibody/peptide inhibitors that block the L-PTC from recognizing intestinal glycan and protein receptors, thereby preventing toxin invasion. The L-PTC could also be exploited for alternative applications. For example, coupling of protein-based therapeutics to a modified non-toxic L-PTC or the HA complex might facilitate drug delivery by enhancing permeability of the intestinal epithelium. Thus, the improved structural understanding of these fascinating macromolecular assemblies informs efforts to treat a deadly toxin and opens opportunities to develop novel therapeutics.

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16••. Lee K, Gu S, Jin L, Le TT, Cheng LW, Strotmeier J, Kruel AM, Yao G, Perry K, Rummel A, et al. Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity. PLoS Pathog.2013;9:e1003690. This is a comprehensive study describing the structure and function of the L-PTC/A. The authors present an atomic model of the L-PTC/A using a combination of X-ray crystallography and single particle EM and 3D reconstruction. Together with biochemical studies, these findings reveal that the HA complex mediates BoNT absorption by binding to host carbohydrates. Furthermore, the authors suggest that carbohydrate receptor mimics could be developed into novel oral inhibitors as preventive countermeasures aganist BoNTs. Abstract]

17••. Benefield DA, Dessain SK, Shine N, Ohi MD, Lacy DB. Molecular assembly of botulinum neurotoxin progenitor complexes. Proc Natl Acad Sci U S A.2013;110:5630–5635. Using EM 3D reconstruction and the known component structures, the authors present the structural models of the fully assembled L-PTC/A and B. They also show that the M-PTC of BoNT/E closely resembles the M-PTC of BoNT/A and BoNT/B. Abstract]

18. Hasegawa K, Watanabe T, Suzuki T, Yamano A, Oikawa T, Sato Y, Kouguchi H, Yoneyama T, Niwa K, Ikeda T, et al. A novel subunit structure of clostridium botulinum serotype D toxin complex with three extended arms. J Biol Chem.2007;282:24777–24783. [Abstract]

19••. Gu S, Rumpel S, Zhou J, Strotmeier J, Bigalke H, Perry K, Shoemaker CB, Rummel A, Jin R. Botulinum neurotoxin is shielded by NTNHA in an interlocked complex. Science.2012;335:977–981. The paper describes the first crystal structure of the M-PTC of BoNT/A, which reveals the molecular mechanism of how NTNHA-A protects BoNT/A through extensive protein-protein interactions. Furthermore, some key residues on BoNT/A that regulate the pH-dependent assembly of the M-PTC were identified. Abstract]

25•. Matsui T, Gu S, Lam KH, Carter LG, Rummel A, Mathews II, Jin R. Structural Basis of the pH-Dependent Assembly of a Botulinum Neurotoxin Complex. J Mol Biol.2014 doi: 10.1016/j.jmb.2014.09.009. This paper reports small-angle X-ray scattering studies on BoNT/A, NTNHA-A, and the M-PTC, which is complementary to the previous X-ray crystallographic studies [19]. It suggests that the assembly of the M-PTC depends on the environmental pH and that BoNT/A adopts a large conformational change that is induced by interacting with NTNHA-A at acidic pH. Abstract] [CrossRef]

26. Sagane Y, Miyashita S, Miyata K, Matsumoto T, Inui K, Hayashi S, Suzuki T, Hasegawa K, Yajima S, Yamano A, et al. Small-angle X-ray scattering reveals structural dynamics of the botulinum neurotoxin associating protein, nontoxic nonhemagglutinin. Biochem Biophys Res Commun.2012;425:256–260. [Abstract]

27. Kouguchi H, Watanabe T, Sagane Y, Sunagawa H, Ohyama T. In vitro reconstitution of the Clostridium botulinum type D progenitor toxin. J Biol Chem.2002;277:2650–2656. [Abstract]

28•. 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. The authors present the structure of the complete HA complex of BoNT/B (HA/B) that was determined by X-ray crystallography. The overall structure of the HA/B is highly similar to the HA/A reported by Lee et al.[16] Abstract]

29. Inoue K, Sobhany M, Transue TR, Oguma K, Pedersen LC, Negishi M. Structural analysis by X-ray crystallography and calorimetry of a haemagglutinin component (HA1) of the progenitor toxin from Clostridium botulinum. Microbiology.2003;149:3361–3370. [Abstract]

31. Nakamura T, Kotani M, Tonozuka T, Ide A, Oguma K, Nishikawa A. Crystal structure of the HA3 subcomponent of Clostridium botulinum type C progenitor toxin. J Mol Biol.2009;385:1193–1206. [Abstract]

32. Yamashita S, Yoshida H, Uchiyama N, Nakakita Y, Nakakita S, Tonozuka T, Oguma K, Nishikawa A, Kamitori S. Carbohydrate recognition mechanism of HA70 from Clostridium botulinum deduced from X-ray structures in complexes with sialylated oligosaccharides. FEBS Lett.2012;586:2404–2410. [Abstract]

33•. Sugawara Y, Yutani M, Amatsu S, Matsumura T, Fujinaga Y. Functional Dissection of the Clostridium botulinum Type B Hemagglutinin Complex: Identification of the Carbohydrate and E-Cadherin Binding Sites. PLoS One.2014;9:e111170. This paper reports the carbohydrate and E-cadherin binding sites on the L-PTC/B, which are similar to that of the L-PTC/A. Abstract]

35. Lee K, Lam KH, Kruel AM, Perry K, Rummel A, Jin R. High-resolution crystal structure of HA33 of botulinum neurotoxin type B progenitor toxin complex. Biochem Biophys Res Commun.2014;446:568–573. Abstract]

36. Inoue K, Fujinaga Y, Honke K, Arimitsu H, Mahmut N, Sakaguchi Y, Ohyama T, Watanabe T, Inoue K, Oguma K. Clostridium botulinum type A haemagglutinin-positive progenitor toxin (HA(+)-PTX) binds to oligosaccharides containing Gal beta1-4GlcNAc through one subcomponent of haemagglutinin (HA1) Microbiology.2001;147:811–819. [Abstract]

37. Kojima S, Eguchi H, Ookawara T, Fujiwara N, Yasuda J, Nakagawa K, Yamamura T, Suzuki K. Clostridium botulinum type A progenitor toxin binds to Intestine-407 cells via N-acetyllactosamine moiety. Biochem Biophys Res Commun.2005;331:571–576. [Abstract]

39. Sagane Y, Hayashi S, Matsumoto T, Miyashita S, Inui K, Miyata K, Yajima S, Suzuki T, Hasegawa K, Yamano A, et al. Sugar-induced conformational change found in the HA-33/HA-17 trimer of the botulinum toxin complex. Biochem Biophys Res Commun.2013;438:483–487. [Abstract]

40••. Lee K, Zhong X, Gu S, Kruel AM, Dorner MB, Perry K, Rummel A, Dong M, Jin R. Molecular basis for disruption of E-cadherin adhesion by botulinum neurotoxin A complex. Science.2014;344:1405–1410. This is the first crystal structure that reveals how the HA complex specifically binds to E-cadherin and disrupts the E-cadherin-mediated intercellular barrier. Furthermore, the authors successfully reconstituted the entire 14-subunit L-PTC/A using recombinant components, which allowed them to perform in vivo studies that directly addressed the physiological roles of host carbohydrates and E-cadherin in oral BoNT intoxication. Abstract]

41. Matsumura T, Jin Y, Kabumoto Y, Takegahara Y, Oguma K, Lencer WI, Fujinaga Y. The HA proteins of botulinum toxin disrupt intestinal epithelial intercellular junctions to increase toxin absorption. Cell Microbiol.2008;10:355–364. [Abstract]

42. Sugawara Y, Matsumura T, Takegahara Y, Jin Y, Tsukasaki Y, Takeichi M, Fujinaga Y. Botulinum hemagglutinin disrupts the intercellular epithelial barrier by directly binding E-cadherin. J Cell Biol.2010;189:691–700. Abstract]

44. Harrison OJ, Jin X, Hong S, Bahna F, Ahlsen G, Brasch J, Wu Y, Vendome J, Felsovalyi K, Hampton CM, et al. The extracellular architecture of adherens junctions revealed by crystal structures of type I cadherins. Structure.2011;19:244–256. Abstract]

45. Harrison OJ, Bahna F, Katsamba PS, Jin X, Brasch J, Vendome J, Ahlsen G, Carroll KJ, Price SR, Honig B, et al. Two-step adhesive binding by classical cadherins. Nat Struct Mol Biol.2010;17:348–357. Abstract]

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49. Jin Y, Takegahara Y, Sugawara Y, Matsumura T, Fujinaga Y. 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.2009;155:35–45. [Abstract]

52. Maksymowych AB, Simpson LL. Structural features of the botulinum neurotoxin molecule that govern binding and transcytosis across polarized human intestinal epithelial cells. J Pharmacol Exp Ther.2004;310:633–641. [Abstract]

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Newswise — Irvine, Calif. – February 27, 2020 – New study reveals potential for developing novel antibody-based antitoxins against botulinum neurotoxins (BoNTs), including the most commonly used, yet most toxic one, Botox.

Published in Cell Reports, the paper is titled, “Structural insights into rational design of single-domain antibody-based antitoxins against botulinum neurotoxins.” Led by Rongsheng Jin, PhD, a professor in the Department of Physiology & Biophysics at the University of California, Irvine, School of Medicine, this paper describes how the team first identified the neutralizing epitopes of six anti-BoNT nanobodies (VHHs) based on their crystal structures, then harnessed the structural findings to rationally design bifunctional nanobodies. Different than ordinary nanobodies, bifunctional nanobodies are composed of two nanobodies that bind simultaneously to the toxins.

Based on a mouse model, their findings revealed the bifunctional nanobodies protected mice with much greater potency than the simple combination of two nanobodies.

“In a nutshell, we establish a platform for structure-based rational design of bifunctional antitoxins against BoNTs,” said Kwok-ho Lam, the first author and a project scientist in the Jin lab. “BoNTs can be misused as a bioweapon and thus have been classified as Tier 1 select agents by the Centers for Disease Control and Prevention, which is why there is urgent need for antitoxins.”

“Currently, the only available antitoxin remedies are polyclonal antibodies from horse or human serum, which have known health risks and are in limited supply. Monoclonal antibodies are still under development,” said Jin. “And, while it isn’t necessarily a cause for worry, the increasingly popular therapeutic uses of BoNT products also create risks of possible botulism resulting from the medical treatments where they are used.”

This study was funded in part by the National Institute of Allergy and Infectious Diseases, the Defense Threat Reduction Agency—Chemical Biological Defense Therapeutics, and the National Institute of General Medical Sciences.

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Newswise — Irvine, Calif. — The Botox toxin has a sweet tooth, and it’s this craving for sugars – glycans, to be exact – that underlies its extreme ability target neuron cells in the body … while giving researchers an approach to neutralize it. A study co-led by Rongsheng Jin, professor of physiology & biophysics at the University of California, Irvine; Min Dong with Boston Children’s Hospital-Harvard Medical School; and Andreas Rummel with the Hannover Medical School in Germany, reveals an important general mechanism by which the pathogen is attracted to, adapts to and takes advantage of glycan modifications in surface receptors to invade motor neurons. Glycans are chains of sugars synthesized by cells for their development, growth, functioning or survival. Results appear June 13 in Nature Structural and Molecular Biology.

“Our findings reveal a new paradigm of the everlasting host-pathogen arms race, where a pathogen develops a smart strategy to achieve highly specific binding to a host receptor while also tolerating genetic changes on the receptor,” Jin said. “And to some extent, this mechanism by which the toxin attacks human is similar to the one that is utilized by some important broad-neutralizing human antibodies to fight viruses, such as dengue viruses and HIV.”

Botulinum neurotoxin A (BoNT/A), commonly known as the Botox toxin, is widely used in a weakened form for treating various medical conditions as well as for cosmetics. The clinical product Botox contains extremely low doses of the toxin and is safe to use. But at higher doses, it can be lethal, and it’s also classified as a potential bioterrorism agent.

The intricate detail of how the Botox toxin recognizes its receptors also reveals novel ways to neutralizing these deadly toxins. “With this new structural information,” Rummel said, “we were able to pinpoint key amino acids in the toxin that are required for binding to sugars, and we found that even mutating a single amino acid is sufficient to abolish the toxicity by more than a million fold.”

Guorui Yao and Kwok-ho Lam at UCI, Sicai Zhang at Harvard, Stefan Mahrhold at the Hannover Medical School, Daniel Stern at Berlin’s Center for Biological Threats & Special Pathogens, Kay Perry at the Argonne National Laboratory in Illinois, and Karine Bagramyan and Markus Kalkum the Beckman Research Institute at the City of Hope in Duarte, Calif., contributed to the study, which was primarily supported by the National Institutes of Health.

About the University of California, Irvine: Currently celebrating its 50th anniversary, UCI is the youngest member of the prestigious Association of American Universities. The campus has produced three Nobel laureates and is known for its academic achievement, premier research, innovation and anteater mascot. Led by Chancellor Howard Gillman, UCI has more than 30,000 students and offers 192 degree programs. It’s located in one of the world’s safest and most economically vibrant communities and is Orange County’s second-largest employer, contributing $4.8 billion annually to the local economy. For more on UCI, visit www.uci.edu.

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CSPG4 is a large highly glycosylated single transmembrane protein (~251 kDa). Its extracellular domain was predicted to contain a signal peptide, two laminin G motifs, and 15 consecutive CSPG repeats1a). Our initial efforts using the recombinant full extracellular domain of human CSPG4 (residues 30–2204, referred to as CSPG4ECD) and TcdB1 holotoxin (VPI10463 strain) were hampered by the structural flexibility of TcdB and CSPG4ECD employing cross-linking mass spectrometry (XL-MS) using the MS-cleavable cross-linker dihydrazide sulfoxide (DHSO)1a, b and Source Data). When forming a complex, acidic residues in TcdB1 and CSPG4ECD that have Cα-Cα distances within 35 Å can be cross-linked by DHSO, and the resulting cross-linked peptides could be identified using multistage mass spectrometry (MSn)

a Schematic diagrams showing the domain structures of TcdB and CSPG4, as well as the domain boundaries for TcdBcore and CSPG4mini used for cryo-EM studies. GTD: glucosyltransferase domain, CPD: cysteine protease domain, DRBD: delivery and receptor-binding domain, CROPs: combined repetitive oligopeptides domain, Hinge: a key fragment between the DRBD and CROPs that mediates structural communications among all four domains of TcdB. CSPG4 is composed of two predicted laminin G domains, 15 CSPG repeats, a transmembrane domain (TM), and a cytosolic region. b The 3.17 Å resolution cryo-EM map of the TcdBcore–Repeat1 complex segmented and colored as shown in a. c Cartoon representation of the structure of the TcdBcore–Repeat1 complex that is shown in similar orientations and color schemes as that in b. d The structure of Repeat1 of CSPG4 with the disulfide bond shown as sticks. e The structure of the TcdBcore–Repeat1 complex was superimposed to TcdB holotoxin (PDB: 6OQ5). The Repeat1-bound TcdB is colored as shown in a and the unliganded TcdB is colored black with its CROPs II–IV omitted for clarity. The TcdB-bound Repeat1 is shown as a green surface model. f Repeat1 triggers local structural changes in the CPD and hinge of TcdB upon binding. For clarity, only residues 569–577 in the CPD and residues 1803–1812 in the hinge are shown in the context of Repeat1 (green surface).

We identified a total of 263 unique DHSO cross-linked peptides of the TcdB1–CSPG4ECD complex (Supplementary Table 1), representing 18 inter-protein and 245 intra-protein (167 in TcdB1 and 78 in CSPG4ECD) cross-links. The intra-molecular cross-links in TcdB1 show good correlations with the crystal structure of TcdB1 holotoxin that we recently reported1c). The rest four pairs of cross-links suggested that the laminin G domains of CSPG4 may adopt flexible conformations and could transiently move within ~35 Å of the CPD or DRBD of TcdB, because the same residues (e.g., E92/E93) in this region of CSPG4 could be cross-linked to amino acids on the CPD and DRBD of TcdB that are 97 Å away from each other (Supplementary Table 1). Guided by the XL-MS results, we analyzed interactions between a number of fragments of TcdB1 and CSPG4 and their biochemical behaviors, and narrowed down a fragment of TcdB1 (residues 1–1967, referred to as TcdBcore) that contains the GTD, CPD, DRBD, and the first unit of CROPs (termed CROPs I), which could robustly bind to an N-terminal CSPG4 fragment composed of two laminin G motifs and first two CSPG repeats (residues 30–764, referred to as CSPG4mini) (Fig. 1a and Supplementary Table 2).

We successfully obtained a stable complex composed of TcdBcore and CSPG4mini, which was used for cryo-EM study (Supplementary Fig. 2a–d). The preliminary data analysis yielded a 3.4 Å resolution structure for the TcdBcore–CSPG4mini complex, which revealed that CSPG4mini binds to a groove in TcdB that is surrounded by the CPD, DRBD, hinge, and CROPs I (Supplementary Fig. 2e, f), which is consistent with our XL-MS studies. 3D variability analysis indicated that the distal region in the DRBD of TcdB and the N-terminal two laminin G motifs of CSPG4mini were highly flexible, which hindered us from obtaining a high-resolution map for de novo model building. Notably, these flexible regions in TcdB and CSPG4 were outside the complex interface. Therefore, we could improve the resolution by using a smaller box size during particle picking to focus on the TcdB–CSPG4 interface. With a focused refinement, we were able to further improve the density map to 3.17 Å resolution that allowed de novo model building for CSPG4, while the TcdB structure was built using the crystal structure of TcdB holotoxin as a model1b, c and Supplementary Fig. 2g, h). Structure determination statistics and representative density maps for the protein complex were shown in Supplementary Table 3 and Supplementary Fig. 3.

The structure of the TcdB–CSPG4 complex reveals that the first CSPG repeat of CSPG4 (termed Repeat1, residues 410–551) is mainly responsible for TcdB binding, while the rest of CSPG4 pointing away from the toxin (Supplementary Fig. 2f). Repeat1 has a compact structure consisting of a four-strand β sheet and 4 short α helices, which are connected by intermittent loops and stabilized by a disulfide bridge (Fig. 1d). Despite its small size, Repeat1 directly interacts with many amino acids that are dispersed across over 1300 residues on the primary sequence of TcdB, including the CPD, DRBD, hinge, and CROPs (Fig. 1b, c). All these TcdB residues converge spatially to form a composite binding site for Repeat1 involving an extensive interaction network and burying a large molecular interface between them (∼2715.5 Å2) (Fig. 2a–c). This unusually complex binding mode, especially the involvement of the CPD, is unexpected, because it was previously believed that the receptor binding of TcdB is carried out by the DRBD and the CROPs

More detailed structural analysis showed that the TcdB-binding surface in Repeat1 could be divided into three subsites (Fig. 2c). The site-1 of Repeat1 (residues 448–457) binds to the CPD via hydrogen bonds, charge–charge interaction, as well as a large patch of hydrophobic interactions (Fig. 2d and Supplementary Table 4). The site-2 of Repeat1 (residues 466–503) binds to the hinge of TcdB involving mainly hydrophobic interaction and two hydrogen bonds, and also interacts with the CROPs I with a hydrogen bond (Fig. 2e and Supplementary Table 4). The site-3 of Repeat1 is composed of two separated areas including residues 457–466 and an additional residue (R527) in a nearby loop. It binds to a composite interface in TcdB, which is composed of residues in the CPD, DRBD, and hinge (Fig. 2f and Supplementary Table 4). CSPG4 is predicted to have 15 N-linked glycosylation site with one in Repeat1 (N427) and a single chondroitin sulfate modification at S995

The overall structure of the CSPG4-bound TcdBcore is similar to the crystal structure of TcdB holotoxin with a root mean square deviation between comparable Cα atoms about 1.06 Å (Fig. 1e)1f). It is worth noting that the hinge is located at a strategic site in TcdB communicating with all four major domains, and the CROPs of TcdB adopts dynamic conformations relative to the rest of the toxin

We further carried out real-time analysis of the kinetics of TcdB–CSPG4 interactions using bio-layer interferometry (BLI). For this study, we first designed a recombinant CSPG4 Repeat1 that is fused to the N-terminus of the Fc fragment of a human immunoglobulin G1 (Repeat1-Fc). Based on the structural modeling, the Fc fragment in Repeat1-Fc does not interfere with TcdB binding, and provide a convenient way for immobilization of Repeat1-Fc to the biosensors. We found that TcdB1 recognized Repeat1-Fc with a high affinity (dissociation constant, Kd ~15.2 nM) (Supplementary Fig. 4a). Notably, Repeat1-Fc binds to TcdB with a relatively slow on-rate (kon ~7.06 × 103 M−1 s−1), which is likely due to organization of multiple structural units in TcdB to form the composite binding site for CSPG4. Nevertheless, once Repeat1 is engaged with TcdB, the complex is very stable as evidence by their slow binding off-rate (koff ~1.08 × 10−4 s−1).

Since TcdB1 and TcdB2 have different primary sequences and pathogenicity, we carried out structure-based sequence analysis between them focusing on the CSPG4-binding site. Remarkably, the key amino acids consisting the composite CSPG4-binding site are nearly identical between TcdB1 and TcdB2, even though these residues scatter across multiple TcdB domains (Fig. 2g). It is worth noting that the hinge region has large sequence variations among TcdB isoforms, and the hypervariable sequences in this region are believed to contribute to differences in toxicity and antigenicity of TcdB2 and other variantsTcdB1 with L1809TcdB2 and V1816TcdB1 with I1816TcdB2. The only other difference is N1850TcdB1 in the CROPs I that forms a hydrogen bond with K503 of CSPG4 is replaced with K1850TcdB2. Nevertheless, our BLI binding studies showed that TcdB2 binds to Repeat1-Fc with a high affinity that is even slightly better than TcdB1 (Kd ~5.4 nM, kon ~8.34 × 103 M−1 s−1, koff ~4.63 × 10−5 s−1) (Supplementary Fig. 4b). Therefore, the three residue substitutions in the CSPG4-binding site are well tolerated in TcdB2. These data demonstrate that the CSPG4-binding mode is conserved between TcdB1 and TcdB2.

We next carried out structure-guided mutagenesis of TcdB1 and CSPG4 to validate the binding interface and to define loss-of-function mutations in TcdB that could selectively abolish CSPG4 binding. We designed and characterized nine mutations of TcdB1 holotoxin, where the key CSPG4-binding residues in the CPD (L563G/I566G, S567E, Y621A, or Y603G), the hinge (D1812G, V1816G/L1818G, or F1823G/I1825G/M1831G), the DRBD (N1758A), or the CROPs I (N1850A) were mutated (Supplementary Fig. 5). These TcdB1 mutants showed reduced binding to HeLa cells expressing endogenous CSPG43a). TcdB-N1758A and N1850A showed the least reduction of binding, suggesting that these two mutations, located in the DRBD and the CROPs respectively, have relatively weaker impact on TcdB–CSPG4 interactions compared with mutations in the CPD or the hinge. We then designed three combinational mutations of TcdB to simultaneously disrupt the anchoring points for CSPG4 in both the CPD and the hinge, including S567E/D1812G, Y603G/D1812G, and S567E/Y603G/D1812G, and found them largely abolished binding of TcdB to cells. Similar results were confirmed using pull-down assays with Repeat1-Fc as the bait and TcdB variants as preys (Supplementary Fig. 6a). We also designed and characterized variants of CSPG4 Repeat1 that carried site-specific mutations in the TcdB-binding interface, including mutations in site-1 (R450G, E448A, W449G, W449D, Q453A, E448A/W449D, R450G/Q453A), site-2 (L497G, L497D, L497G/D498G), and site-3 (D457G, R464A/S466G) (Supplementary Fig. 7). These mutations effectively disrupted the binding of TcdB holotoxin to Repeat1 based on pull-down assays (Supplementary Fig. 6b).

a The indicated TcdB mutants were tested for binding to cells. Purified WT and mutated TcdB (10 nM) were incubated with WT or CSPG4−/− HeLa cells. Cells were washed three times by PBS, harvested, and cell lysates were analyzed by immunoblot detecting TcdB. Actin served as a loading control. The sensitivity of CSPG4−/− (b) and WT (c) HeLa cells to mutated TcdB was examined using the standard cytopathic cell-rounding assay. Error bars indicate mean ± sd (n = 3 biologically independent experiments). d The ratios of CR50 values on CSPG4−/− vs. WT HeLa cells from b and c were calculated and plotted, reflecting the fold-of-change in reduction of toxicity on CSPG4−/− cells compared with WT cells. n = 3 for all groups. The upper and lower bounds of boxes indicate the maximum and minimum values of each group. The middle lines indicate the median values of each group. p values by t-test: *p ≤ 0.05.

We further examined how these TcdB mutations effect CSPG4-mediated cytopathic toxicity at functional levels using standard cell-rounding assays, where TcdB entry would inactivate Rho GTPases and cause the characteristic cell-rounding phenotype50), which is utilized to compare the potency of TcdB variants on the wild-type (WT) HeLa cells that express both CSPG4 and FZDs or the CSPG4 knockout (KO) HeLa cells. As shown in Fig. 3b, all 12 mutant TcdB1 induced cell-rounding with potencies similar to TcdB1 on CSPG4 KO cells, demonstrating that these mutations were properly folded and did not affect FZD-mediated binding and entry of toxins. In contrast, these mutant toxins showed various reduced potencies on WT HeLa cells compared with TcdB1 (Fig. 3c). More specifically, WT TcdB1 showed over 600-fold reduced toxicity on CSPG4 KO cells compared with WT cells, while the toxicity of TcdB1 variants carrying L563G/I566G, D1812G, V1816G/L1818G, F1823G/I1825G/M1831G, and the three combinational mutations were similar on CSPG4 KO cells and WT cells (CR50 ratio ~1.1–1.3), demonstrating that these mutations effectively and selectively eliminated CSPG4-mediated toxicity on cells (Fig. 3d).

Given our extensive structural, in vitro, and ex vivo data demonstrating the role of CSPG4 as a TcdB receptor, we sought to determine the contribution of CSPG4 to TcdB1 and TcdB2 pathogenicity and its relationship with FZD in vivo using two complementary approaches that were custom designed for TcdB2 and TcdB1, respectively.

We next carried out histological analysis of cecum and colon tissues. There was bloody fluid accumulation in tissues dissected from WT mice after infection, whereas there was much less fluid accumulation in tissues from CSPG4 KO mice (Fig. 4a). We further carried out histological analysis with paraffin-embedded cecum tissue sections (Fig. 4b), which were scored based on disruption of the epithelium, hemorrhagic congestion, submucosal edema, and inflammatory cell infiltration, on a scale of 0–3 (normal, mild, moderate, or severe, Fig. 4c). Infection induced extensive disruption of the epithelium and inflammatory cell infiltration, as well as severe hemorrhagic congestion and mucosal edema on WT mice (Fig. 4c). CSPG4 KO mice showed only moderate levels of epithelial damage and inflammatory cell infiltration, and mild to no hemorrhagic congestion and submucosal edema (Fig. 4b, c). Furthermore, TcdB2 induced extensive loss of tight junction in the cecum epithelium from WT mice based on immunofluorescence staining for a tight junction marker Claudin-3, while it was largely intact in CSPG4 KO mice (Fig. 4d). We observed similar results when we carried out infection experiments using a ten-fold lower dose of C. difficile spores (1 × 104), which did not result in death of mice and thus allowed us to harvest cecum tissues 90 h after infection (Supplementary Fig. 8c–e). Analysis of feces indicated similar levels of C. difficile colonization and toxin titer in WT and CSPG4 KO mice (Supplementary Fig. 8c). Taken together, these results demonstrated that CSPG4 is a major receptor for the epidemic TcdB2 in vivo. The residual toxicity of TcdB2 in CSPG4 KO mice indicates that TcdB2 may have unknown low affinity receptor(s) that remains to be further evaluated.

a Three groups of infection experiment were performed: mock to WT (n = 4); M7404, tcdA− to WT mice (n = 8); and M7404, tcdA− to CSPG4−/− mice (n = 9). The representative cecum and colon of infected mice that were harvested at 48 h. The harvested cecum was processed with hematoxylin and eosin staining (scale bar represents 100 µm, mock n = 4, C. difficile to WT n = 4, C. difficile to CSPG4−/− n = 5) (b), scored based on inflammatory cell infiltration, hemorrhagic congestion, epithelial disruption, and submucosal edema (c), and subjected to immunofluorescence staining by epithelial cell junction marker Claudin-3 (scale bar represents 50 µm, mock n = 3, C. difficile to WT n = 3, C. difficile to CSPG4−/− n = 3) (d). In c, error bars indicate mean ± SEM (mock n = 4, C. difficile to WT n = 4, C. difficile to CSPG4−/− n = 5). p values were calculated by post hoc analysis of a one-way ANOVA using Holm-Sidak’s test for multiple comparisons: ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. The exact p values are presented in the accompanying source data.

TcdB1 can be simultaneously bound by CSPG4 and FZD as demonstrated by our cryo-EM structure of the TcdB–CSPG4 complex and the crystal structure of a TcdB–FZD complex, which was confirmed by a pull-down experiment (Supplementary Fig. 8f)5a). To investigate the relationship of these two receptors for TcdB1, we resorted to three structure-based rationally designed TcdB1 mutants as molecular tools, which carry site-specific mutations to selectively knockout its binding capacity to CSPG4, FZD, or both. Based on the mutagenesis studies described above, we chose to use TcdBS567E/Y603G/D1812G as a representative CSPG4 binding deficient TcdB mutant (TcdBCSPG4−). We previously already developed a FZD-binding deficient TcdB variant that carries mutations in the FZD-binding site (TcdBGFE)FZD−/CSPG4−).

a A structural model of TcdB holotoxin with CSPG4 and FZD bound at two independent sites. The model is built based on superposition of the structures of TcdB1 holotoxin (PDB: 6OQ5), the TcdB–FZD complex (PDB: 6C0B), and the TcdB–CSPG4 complex (this work). b–d The indicated TcdB mutants or the control PBS was injected into the cecum of CD1 mice in vivo. The cecum tissues were harvested 6 h later and subjected to histological analysis with representative images (scale bars represent 100 µm, PBS n = 4, TcdB n = 5, TcdBGFE n = 5, and TcdBFZD−/CSPG4− n = 5, TcdBCSPG4− n = 5) (b), immunostaining analysis for the tight junction marker Claudin-3 (scale bars represent 50 µm, PBS n = 3, TcdB n = 3, TcdBGFE n = 3, and TcdBFZD−/CSPG4− n = 3, TcdBCSPG4− n = 3) (c), and pathological scores (error bars indicate mean ± SEM, PBS n = 4, TcdB n = 5, TcdBGFE n = 5, and TcdBFZD−/CSPG4− n = 5, TcdBCSPG4− n = 5) (d). p values were calculated by post hoc analysis of a by one-way ANOVA using Holm-Sidak’s test for multiple comparisons: ****p ≤ 0.0001, ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05. The exact p values are presented in the accompanying source data.

We analyzed the toxicity of these TcdB1 mutants in comparison with the WT toxin by directly inject them into the mouse cecum5b–d and Supplementary Fig. 8g). Both TcdBGFE and TcdBCSPG4− showed greatly reduced potency, with no significant difference between them: both showed modest levels of inflammatory cell infiltration and submucosal edema, and mild to normal levels of disruption of epithelium, tight junction, and hemorrhagic congestion. TcdBFZD−/CSPG4− showed further reduced toxicity, with minimal levels of disruption to cecum tissues under our assay conditions (Fig. 5d and Supplementary Fig. 8g). These results demonstrate that FZDs and CSPG4 act as independent receptors in TcdB1 pathogenesis in vivo.

Bezlotoxumab is the only FDA-approved therapeutic antibody against TcdB, and a prior study suggested that bezlotoxumab reduced binding of TcdB to CSPG4 in vitro in immunoprecipitation assays9a)6a)6b). Since CSPG4 binds TcdB by simultaneously interacting with the CPD, DRBD, hinge, and CROPs, bezlotoxumab binding may reorient the CROPs relative to the rest of TcdB and compress the CSPG4-binding groove, thus preventing CSPG4 binding in an allosteric manner (Fig. 6b).

a A structure model showing the binding of CSPG4 and bezlotoxumab (PDB: 4NP4) in TcdB holotoxin (PDB: 6OQ5). TcdB holotoxin and CSPG4 Repeat1 are showing as surface models with the GTD, CPD, DRBD, CROPs, and CSPG4 Repeat1 colored in pink, blue, orange, cyan, and green, respectively. The two Fab fragments of bezlotoxumab are shown as cartoon models and colored blue and purple. E1 and E2 indicate the epitope-1 and epitope-2 for bezlotoxumab in TcdB. A