Structural Elucidation of the Clostridioides Difficile Transferase Toxin Reveals a Single-Site Binding Mode for the Enzyme

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Structural elucidation of the Clostridioides difficile transferase toxin reveals a single-site binding mode for the enzyme

Michael J. Sheedloa,b, David M. Andersona,b, Audrey K. Thomasa, and D. Borden Lacya,b,1

aDepartment of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232; and bThe Veterans Affairs Tennessee Valley Healthcare System, Nashville, TN 37232

Edited by Stephen C. Harrison, Boston Children’s Hospital, Boston, MA, and approved February 5, 2020 (received for review November 21, 2019) Clostridioides difficile is a Gram-positive, pathogenic bacterium described a synergistic effect between CDT and TcdA/B which and a prominent cause of hospital-acquired diarrhea in the United enhanced innate immune signaling and dampened the eosinophil States. The symptoms of C. difficile infection are caused by the response (15). Although these two mechanisms are seemingly activity of three large toxins known as toxin A (TcdA), toxin B unrelated, it is possible that the presence of CDT is not restricted (TcdB), and the C. difficile transferase toxin (CDT). Reported here to either of these functions and that the combination of these is a 3.8-Å cryo–electron microscopy (cryo-EM) structure of CDT, a two events leads to increased disease severity during infection. bipartite toxin comprised of the proteins CDTa and CDTb. We The structure of each component of CDT has been previously observe a single molecule of CDTa bound to a CDTb heptamer. determined. CDTa has been characterized by X-ray crystallog- The formation of the CDT complex relies on the interaction of an raphy and is known to consist of two domains: a pseudo-ADP N-terminal adaptor and pseudoenzyme domain of CDTa with six ribosyltransferase (pADPRT) domain and an ADP ribosyltransferase subunits of the CDTb heptamer. CDTb is observed in a preinsertion (ADPRT) domain. The ADPRT domain is the enzymatic com- state, a conformation observed in the transition of prepore to ponent of CDT and functions by modifying actin, leading to a β -barrel pore, although we also observe a single bound CDTa in distinct cytopathic cell-rounding phenotype (Fig. 1A) (16). The β the prepore and -barrel conformations of CDTb. The binding pADPRT and ADPRT domains are similar in sequence and

interaction appears to prime CDTa for translocation as the adaptor structure, and both are conserved among members of the Iota MICROBIOLOGY subdomain enters the lumen of the preinsertion state channel. family of binary toxins, as well as the related C2 toxin produced These structural observations advance the understanding of how by Clostridium botulinum (SI Appendix, Fig. S1 A and B). Because a single protein, CDTb, can mediate the delivery of a large enzyme, these two domains are so similar it has been hypothesized that CDTa, into the cytosol of mammalian cells. the structure of CDTa arose from an ancestral gene duplication event, although the benefit of conserving this architecture binary toxin | cryo-EM | Iota toxin | Clostridium throughout evolution remains unclear (7, 12). The pore-forming component of CDT is CDTb, a five-domain protein that forms C lostridioides difficile is a pathogenic, Gram-positive bacte- oligomeric pores to facilitate the transfer of CDTa into the host rium that forms a hardy spore, which can persist through cytoplasm. The five domains of CDTb are called D1–D3, D3′,and extreme conditions and can be difficult to eradicate (1). The spores are present in the environment and are able to infect Significance humans via the fecal–oral route. In situations where the gut microbiota has been disrupted, most frequently through the use Clostridioides difficile Clostridium difficile of broad-spectrum antibiotics, C. difficile can colonize and pro- (formerly ) is the lead- liferate in the colon to cause disease. C. difficile infection (CDI) ing cause of hospital-acquired diarrhea in the United States. The C. difficile has become the leading cause of hospital-acquired diarrhea in pathology resulting from infection has been attributed the United States and leads to billions of dollars in additional to the activity of up to three secreted toxins known as toxin A C. difficile healthcare expenditures each year (2). The disease state is me- (TcdA), toxin B (TcdB), and the transferase toxin (CDT). diated by two large, homologous toxins termed toxin A (TcdA) Reported here is the near-atomic resolution structure of CDT, a and toxin B (TcdB) (3). Both TcdA and TcdB belong to the large bipartite toxin that is made up of two components called CDTa glucosylating family of toxins and function by modifying small Rho and CDTb. Our structure highlights a unique mode of toxin as- family GTPases, leading to cytoskeletal disruption and cell death sembly that is distinct from the previously characterized anthrax (4–6). Although these two toxins can alone elicit all known toxin. The orientation we observe appears to prime CDT for symptoms associated with CDI, some of the most problematic delivery into the host by placing the N-terminal domain of CDTa clinical strains also produce a third toxin termed the C. difficile at the entrance to the pore. transferase toxin (CDT, or binary toxin) (7–9). – Author contributions: M.J.S., D.M.A., and D.B.L. designed research; M.J.S., D.M.A., and CDT is an A B-type toxin and a member of the Iota family of A.K.T. performed research; M.J.S. and D.M.A. contributed new reagents/analytic tools; binary toxins. It is comprised of two polypeptide chains termed M.J.S., D.M.A., and D.B.L. analyzed data; and M.J.S., D.M.A., A.K.T., and D.B.L. wrote CDTa, which is the enzymatic component, and CDTb, which the paper. functions as the cell-binding, pore-forming, and delivery appa- The authors declare no competing interest. ratus (10–12). It is currently unclear what role CDT plays during This article is a PNAS Direct Submission. C. difficile pathogenesis, although the prevalence of it in the so- Published under the PNAS license. “ ” called hypervirulent strains suggests that it may be involved in Data deposition: The data described in this publication are made available through the promoting severe infection (9, 13). An initial study on this topic Electron Microscopy Data Bank (EMDB accession code 21016) and the Protein Data Bank noted the formation of microtubule protrusions from colonic (PDB accession code 6V1S). epithelial cells intoxicated by CDT (14). These protrusions are 1To whom correspondence may be addressed. Email: [email protected]. thought to be a direct result of actin depolymerization and have This article contains supporting information online at https://www.pnas.org/lookup/suppl/ been shown to interact with C. difficile, potentially allowing the doi:10.1073/pnas.1920555117/-/DCSupplemental. bacterium to persist at the site of infection. A second study First published March 2, 2020.

www.pnas.org/cgi/doi/10.1073/pnas.1920555117 PNAS | March 17, 2020 | vol. 117 | no. 11 | 6139–6144 Downloaded by guest on September 27, 2021 Fig. 1. Structure of the Clostridioides difficile transferase toxin, or CDT. (A) CDT is a bipartite toxin that is comprised of two polypeptide chains termed CDTa and CDTb. CDTa consists of two domains called the pseudo-ADP ribosyltransferase domain (pADPRT, orange) and the ADP ribosyltransferase domain (ADPRT, red). Encoded within the N terminus of each domain is an adaptor (termed either A1 or A2 and shown in yellow and pink, respectively). CDTb consists of five domains termed D1–D3, D3′ and D4. (B) In our map of the CDT toxin, we have resolved one molecule of CDTa and one CDTb heptamer, colored as in A. On the left is the full map, and on the right is a split view showing the positioning of CDTa within the heptamer. (C) CDTa is centered on top of the CDTb heptamer and makes contact with six out of the seven chains of CDT. (CDTa is represented as a density map and is shown in the same color scheme as indicated in A, and CDTb is shown as a cartoon; chains are denoted A–G.) Protomers that are involved in mediating this interaction are highlighted in red. (D) The structure of the N-terminal adaptor with the helices numbered as shown. (E) The N-terminal helix of CDTa, α1, is buried within the interior of the CDTb heptamer and ∼25 Å above the φ-clamp (shown in green, F).

D4 (Fig. 1A) (17). These domains are responsible for preventing (Fig. 1B). When modeling this interaction, we were able to build premature oligomerization (D1), forming the oligomerization in- almost all of CDTa, although the resolution is markedly lower terface and β-barrel pore (D2/D3), interacting with glycans (D3′), within the ADPRT domain (SI Appendix,Fig.S4A). CDTb was and binding to the host cell receptor (D4). In addition to con- modeled as its first three domains: D1, D2, and D3 (residues 213– taining the pore-forming structure, the D2 domain contains an 556). Although D3′, D4, and the LSR receptor ectodomain were important regulatory feature known as the φ-clamp. This structure present within this sample, they were not observed in the map and consists of seven phenylalanines positioned in a ring at the en- thus could not be modeled. We observed and modeled two cal- trance to the pore and was first characterized in the context of the cium ions into D1, similar to those which were observed in PA related protein, anthrax toxin protective antigen (PA) (18). The (23). These calcium ions are present in all seven chains of the φ-clamp is needed to support cargo translocation and has been CDTb heptamer and are coordinated by a mixture of aspartic acid/ shown to be important for CDT function (17, 18). glutamic acid side chain carboxylates as well as backbone carbonyls The mechanism of CDT intoxication shares many similarities (SI Appendix,Fig.S5). with other Iota family toxins (SI Appendix, Fig. S2A), as well as CDTa is positioned at the “top” and center of the CDTb anthrax toxin (SI Appendix, Fig. S2B) and botulinum C2 (19). heptamer with an interface that is formed using six out of the CDT uses a cell surface receptor known as the lipolysis-stimulated seven chains of CDTb (as determined from ePISA, Fig. 1C) (24). lipoprotein receptor (LSR) to engage host cells (20). Upon lo- Few structural changes occur within CDTa upon complex forma- calization to the membrane, CDTb is cleaved, presumably by cell- tion, as indicated by a low backbone rmsd (1.45 Å) when comparing membrane associated proteases, leading to the formation of a the CDTa crystal structure (Protein Data Bank, PDB 2WN5) and heptameric prepore (21, 22). These prepores are able to interact the cryo-EM structure reported here. The interactions that con- with CDTa, and the complexes are internalized by endocytosis. tribute to CDT toxin formation are numerous and constitute a The acidification of the endosome is thought to trigger CDTb pore relatively large buried surface area of 1,577 Å2. Although we observe formation and the delivery of CDTa through the pore. Previous six chains forming the interface with CDTa, most of the interaction is work on oligomeric CDTb yielded structures in four distinct states mediated by only four chains of CDTb, which have a combined that depict the transition of CDTb from soluble prepore to pre- buried surface area of 1,451 Å2. Key to maintaining this interaction insertion state and then from a partial β-barrel to a full β-barrel are a number of hydrogen bonds and electrostatic interactions fa- pore (17). Three of these states were obtained from particles that cilitated by residues within the D1 (D217, D218, D220, N225, and consisted of double heptamers: a complex of a prepore and a full Y274) and D2 (S492, S493, and Q495) domains of CDTb (SI Ap- β-barrel pore and a complex of two heptamers in the partial pendix,Fig.S6). Although fewer residues of the D2 domain contribute β-barrel state. The preinsertion state was obtained in the presence to this interaction, the loop containing S492, S493, and Q495 (referred of receptor ectodomain and was visualized as single heptameric to here as the SS loop for the two serines) is observed making particles. Since the single heptamers are likely to be more physi- contact with CDTa in three different chains of the CDTb heptamer. ologically relevant, we initiated cryo–electron microscopy (cryo- The SS loop of CDTb likely constitutes the major structural feature EM) studies to probe the structure of CDTb in the preinsertion that is required for CDTa binding. Intriguingly, these serine residues state when bound to CDTa. are not invariable in similar toxins and are substituted by either as- paragine or glutamine at either position (SI Appendix,Fig.S1C). Results A number of residues from CDTa contribute to the interac- One Molecule of CDTa Interacts with One CDTb Oligomer. We have tion with CDTb, and all are within the pADPRT domain or its solved the structure of CDT to 3.8 Å using cryo-EM with local N-terminal adaptor (Fig. 1D). Many of the residues that contribute resolutions of this reconstruction reaching to 3.7 Å (SI Appendix, to this interaction are conserved throughout the Iota family of Figs. S3 and S4 A and B). Despite observing a preferred orien- toxins (SI Appendix,Fig.S1A). In the case of C. perfringens and C. tation of particles within our cryo-EM conditions, views through- botulinum, there are crystal structures of homologous enzymatic out the entire Euler sphere were sampled during refinement (SI proteins that indicate these residues are located in nearly identical Appendix, Fig. S4C). The resulting map contains one molecule of positions, suggesting they may also contribute to toxin complex CDTa bound to one CDTb heptamer in a “preinsertion” state formation (SI Appendix,Fig.S1D) (16, 25, 26). Contact residues

6140 | www.pnas.org/cgi/doi/10.1073/pnas.1920555117 Sheedlo et al. Downloaded by guest on September 27, 2021 from the adaptor include R33 and R40, which interact with the SS CDTb oligomer was 2 nM, and the CDTa Δα1–α3truncation loop, and D93, which forms an interaction with the D1 domain. bound similarly with a binding affinity of 13 nM (Fig. 3 A and B). This arrangement places the first helix of CDTa directly in the No binding was observed between CDTa Δα1–α4andCDTb center of the CDTb oligomer pointing it down toward the fully (Fig. 3 C and D). As an alternative approach we used Microscale formed φ-clamp. The distance between the end of helix 1 and the Thermophoresis (MST) and also observed clear binding (10–20 nM) φ-clamp–forming residue F455 is ∼25 Å (Fig. 1 E and F). We were with CDTa and CDTa Δα1–α3 and no binding with CDTa Δα1–α4 unable to model 10 amino acids at the N terminus of CDTa which (SI Appendix,Fig.S7A–C). To ensure that the truncations did are suspected to be dynamic and unstructured within the interior not result in protein instability, we assayed the thermal stability of the CDTb heptamer. and determined a melting temperature of ∼52 °C for all three forms of CDTa (SI Appendix,Fig.S7D–F). Because CDTa Δα1–α3 CDTa Interacts Similarly with All States of CDTb. The finding that retains interaction, we sought to understand how this truncation one molecule of CDTa engages a CDTb heptamer was not an- might affect the cytopathic cell-rounding effects observed during ticipated as it is distinctly different from what has been observed CDT intoxication. Using Caco-2 colonic epithelial cells, we observed in anthrax toxin PA, which is known to form prepore oligomers robust changes in the actin cytoskeletal structure when wild-type of either seven or eight protomers and can bind either three or CDTa was incubated with CDTb, but no activity was observed four edema factor / lethal factor (EF/LF) proteins, respectively. when CDTa Δα1–α3 or CDTa Δα1–α4 was used (Fig. 3 A–C). EF/LF binding occurs at a site formed from neighboring PA protomers and is located further from the center, such that Discussion multiple molecules can bind the prepore simultaneously. Since The finding that one molecule of CDTa engages a CDTb hep- CDTa was bound to CDTb in a preinsertion state, we considered tamer was not anticipated as it was previously hypothesized that the possibility that CDTa had moved from an outer site into the CDT would assemble with a stoichiometry similar to that of center following the conversion of prepore to the preinsertion anthrax toxin. Anthrax toxin is comprised of three different state. We thus sought to confirm that CDT retained this unique polypeptides known as protective antigen (PA), edema factor binding mode throughout all stages of pore maturation by ana- (EF), and lethal factor (LF). PA is known to form oligomers of lyzing the binding of CDTa to large oligomeric CDTb particles either seven or eight protomers which are believed to simulta- that we have previously shown consist of CDTb in a prepore, neously engage up to four cargo at a time. Structural analysis of partial β-barrel, and pore form (17). We used negative stain EM the PA–LF complex has shown that LF binds at the interface of β two adjacent PA protomers, which form a cleft that is referred to

to visualize CDTa bound to the prepore, partial -barrel, and full MICROBIOLOGY β-barrel forms of CDTb and noted that CDTa was bound in the as the α-clamp. This clamp is a portion of PA that directs binding center each time (Fig. 2 A and B). We also overlaid the models of the EF/LF N terminus into the lumen of the prepore (27). of the CDT complex in the prepore, partial β-barrel, and pore From our initial structural analysis, it was clear that this feature states (PDB 6O2M, 6O2N) with the CDTa–CDTb complex was not retained in CDTb due to large differences in the surface structure and noted that all structural features required for electrostatics between CDTb and PA as well as between EF/LF binding remained in similar positions (Fig. 2 C and D) (17). and CDTa (17). From the structure presented here, we now know that CDT does not possess a homologous structure to the In Vitro Binding Experiments Reveal Portions of the Adaptor Necessary α-clamp, but instead uses the SS loop to engage CDTa. This for Binding and Translocation. To assess the role of the CDTa binding mode is likely the result of an adaptation within the Iota adaptor helices in CDTb binding, we created CDTa mutants family of toxins that arose from the requirement of these toxins that lacked helices α1–α3(CDTaΔα1–α3) and α1–α4(CDTa to translocate a different cargo. Although it is curious that this Δα1–α4). Biolayer Interferometry (BLI) was used to determine loop is not invariable within the Iota family of toxins, we suspect the binding affinities of these proteins and full-length CDTa that substitution of S492 and S493 by Q/N in other Iota toxins is for CDTb. The binding constant (Kd) of full-length CDTa for tolerated, as all can form hydrogen-bonding interactions. It is

Fig. 2. CDTa interactions as determined for all observable forms of oligomeric CDTb. (A) Negative stain analysis of large CDTb oligomeric particles that contain prepores and β-barrel structures reveals that CDTa binds in a similar orientation to what is observed in the preinsertion state as indicated by the black (no CDTa) and white (with CDTa) arrows. (B) A similar approach was taken with large CDTb particles that contained only partial β-barrels on both ends, indicating that CDTa retains the same binding mode in these particles as well, indicated by the black (no CDTa) and white arrows (with CDTa). (C) CDTa contact residues from the CDTb preinsertion state are colored with the SS loop in orange and residues from the D1 domain in yellow. The positions of these contact residues are similar when CDTb adopts the prepore, partial β-barrel, and full β-barrel conformations. (D) We note that no large structural changes occur within CDTb upon binding to CDTa as indicated when all of these models are overlaid.

Sheedlo et al. PNAS | March 17, 2020 | vol. 117 | no. 11 | 6141 Downloaded by guest on September 27, 2021 previously characterized contribution of the pADPRT to complex formation (28). Although no additional interactions are formed by residues within the ADPRT domain, residues within the ADPRT that are homologous to interacting residues within the pADPRT are mostly conserved (Fig. 4). The notable ex- ceptions to this conservation are residues within the N-terminal helical subdomains we have termed “adaptors.” Residues that contribute to this interaction within the pADPRT adaptor (R33, R40, and D93) are not conserved within the ADPRT adaptor (D237, P244, and P295, respectively). It is likely that these substitutions would preclude the ADPRT domain from directly interacting with CDTb in the absence of the pADPRT adaptor, giving rise to an explanation as to why the pADPRT adaptor domain was retained throughout evolution. This does not account for the conservation of the pADPRT in its entirety, as many of the residues that contribute to this interaction from the pseudocata- lytic domain are conserved between the two domains. However, this finding might suggest that the noncatalytic cleft of the pADPRT plays a different role during the course of infection. Our analysis of adaptor truncations shows that although CDTa Δα1–α3 retains interaction with CDTb, it is no longer able to evoke a cytopathic response. This is likely due to a translocation defect in the CDTa Δα1–α3 construct as the N terminus would not be long enough to engage the φ-clamp. In the future, it will be in- teresting to probe the effect of finer truncations on the kinetics and efficiency of cargo delivery and to determine the role that specific residues within the adaptor domain play during CDTa translocation. Materials and Methods Construct Preparation and Protein Purification. An overexpression plasmid

encoding CDTa residues 17–420 and N-terminal His6 and maltose-binding protein tags (pBL926) was transformed into BL21–RIL cells and grown to an

optical density at 595 nm (OD595)of0.4–0.8. Protein synthesis was induced with

Fig. 3. Contribution of the adaptor domain to both binding and translocation. (A) The dissociation constant of wild-type CDTa for the CDTb heptamer was determined by biolayer interferometry. Activity of wild-type CDTa was confirmed in cytopathic assays in which we observed cell rounding when both CDTa and CDTb were present (DAPI staining is shown in blue, phalloidin staining is shown in green). We also tested for binding and the cytopathic cell-rounding phenotype for two CDTa truncations, CDTa Δα1–α3 (shown in B) and CDTa Δα1–α4 (shown in C). The binding constants derived from biolayer interferometry experiments are denoted in D.

likely that more substantial substitutions within the SS loop would not be tolerated. The analysis that we present here demonstrates that CDTa can bind multiple forms of CDTb and that the structural features that are required to mediate CDTa interaction change little during this process. This observation would then suggest that pore formation alone is not enough to stimulate CDTa translocation. We have previously noted that the mechanism for CDTb β-barrel formation does not rely on acidification alone to drive this process. Because we now suggest that CDTa translocation is not stimulated by the structural rearrangements that accompany pore formation, it is conceivable that CDT traffics into the endosome as a fully formed pore. In this proposed model, it would then be Fig. 4. Residues from the pADPRT that contribute to complex formation. CDTa translocation, and not pore formation, that is stimulated by (A) Residues from CDTa that make contact with CDTb are all located within acidification. This mechanism would reconcile previous observa- the pADPRT domain. These residues are scattered between the N-terminal adaptor domain (shown in yellow) and the pseudoenzymatic cleft (shown in tions that have shown that acidification is required to induce cy- orange). A high degree of sequence and structural homology exists between topathic cell rounding as observed in the Iota toxin family (10). the pADPRT and ADRPT as shown in B. The residues which mediate in- The residues of CDTa that contribute to complex formation teraction with CDTb are mostly conserved between the enzymatic clefts but are all positioned within the pADPRT domain, confirming the are different within the adaptor domains (highlighted in gray).

6142 | www.pnas.org/cgi/doi/10.1073/pnas.1920555117 Sheedlo et al. Downloaded by guest on September 27, 2021 a final concentration of 250 μMisopropylβ-D-1-thiogalactopyranoside (IPTG), (PDB 2WN4) into the density map using UCSF Chimera (16, 33). The model and the cultures were incubated at 18 °C for 16 to 18 h. The cells were was then adjusted in COOT and refined in phenix.real_space_refine with harvested by centrifugation and resuspended in 20 mM Hepes pH 8.0, secondary structure and Ramachandran restraints applied during each round 100 mM NaCl, 10% glycerol before being disrupted by an Emulsiflex of refinement (34–36). The resulting model quality was also assessed by microfluidic homogenizer. The lysed cellular suspension was cleared by cen- model-map Fourier shell correlation (SI Appendix, Fig. S4D). All software trifugation and applied to chelating Sepharose resin charged with nickel. The that was used for model building and analysis was accessed through the resin was washed with 5 column volumes 20 mM Hepes pH 8.0, 100 mM NaCl, SBGrid consortium (37). 10% glycerol before eluting CDTa with 20 mM Hepes pH 8.0, 100 mM NaCl, 10% glycerol, 150 mM Imidazole. The eluate was dialyzed into 20 mM Tris Biolayer Interferometry. Kinetic binding analysis between immobilized CDTb pH 7.0, 100 mM NaCl, 10% glycerol, and the affinity tags were concurrently prepore and solution phase CDTa was conducted using an Octet RED96 removed with PreScission Protease. The sample was then further purified by system. Streptavidin sensor tips were first subjected to a 30-s sensor check step cation exchange chromatography and size exclusion chromatography. All CDTa truncations were prepared using the QuikChange method with each construct in loading buffer (20 mM Hepes, pH 8.0, 100 mM NaCl), followed by a 10-min confirmed by Sanger sequencing. Primers that were used to construct CDTa Δα1– loading step with loading buffer in the presence of 30 nM biotinylated α3 (pBL946) and CDTa Δα1–α4 (pBL947) are noted in SI Appendix,TableS1.Each oligomer. Sensor tips were then preblocked for 5 min in kinetic buffer (20 mM truncation was purified following a virtually identical protocol as that noted Hepes, pH 8.0, 100 mM NaCl, 0.1% BSA [RPI, A30075], and 0.005% tween-20). above. The thermostability of each construct was assayed at 1 μMwitha CDTa association was examined via a 60-s binding step in kinetic buffer, Nanotemper Tycho to ensure all constructs were correctly folded. followed by a 20-min dissociation phase. After reference sensor subtraction, A construct containing the LSR ectodomain with an N-terminal human data were fit to a 1:1 binding model with binding affinity estimation cal-

serum albumin secretion signal and C-terminal His6 tag (pBL839) was culated from the response signals. transfected into ExpiCHO cells following manufacturer’s instructions. Protein production was allowed to proceed for 8 d before the cultures were har- Microscale Thermophoresis. The CDTb preinsertion state was purified as de- vested. The cellular suspension was removed by centrifugation and the su- scribed above. This sample was then labeled with NHS reactive Alexa-647 dye pernatant applied to chelating Sepharose resin charged with nickel. The at a 1:4 molar ratio for 1 h at room temperature. Excess dye was quenched resin was washed with 5 column volumes 20 mM Hepes pH 8.0, 100 mM NaCl with 1 M Tris pH 8.0 and subsequently removed over a PD10 desalting column and eluted with 20 mM Hepes pH 8.0, 100 mM NaCl, 150 mM Imidazole. LSR and by size exclusion chromatography. Unlabeled CDTa was then serially was further purified by size exclusion chromatography. diluted over 16 reactions starting with a concentration of 2 μM. Labeled CDTb A CDTb construct containing residues 43–876 with an N-terminal His tag 6 preinsertion state was then added into each reaction to a final concentra- (pBL870) was transformed into BL21–RIL cells and grown to an OD of 595 tion of 50 nM. The samples were loaded onto a NanoTemper Monolith and 0.4–0.8. Protein production was initiated with 250 μM IPTG, and the cultures binding assessed with MST power set to medium. All data were analyzed in were further incubated at 18 °C for 16–18 h. After incubation the cells were Palmist (38). harvested via centrifugation and resuspended in 20 mM Hepes pH 8.0, MICROBIOLOGY 100 mM NaCl. The cells were disrupted with an Emulsiflex microfluidic homogenizer and the resulting suspension cleared by centrifugation. The Cytopathic Cell-Rounding Assay. Purified nicked-CDTb monomer and CDTa resulting supernatant was applied to chelating Sepharose resin charged with were mixed to a final concentration of 1 nM CDT toxin (1 nM CDTa + 7nM nickel and washed with 5 column volumes 20 mM Hepes pH 8.0, 100 mM CDTb monomer). The stock solution was diluted into EMEM medium sup- NaCl before eluting CDTb with 20 mM Hepes pH 8.0, 100 mM NaCl, 150 mM plemented with 20% FBS before being applied to Caco-2 cells that had been Imidazole. CDTb was further purified by anion exchange chromatography grown to ∼80% confluency on a glass cover slide. The cells were then further

and size exclusion chromatography. CDTb oligomerization was initiated by incubated at 37 °C and 5.0% CO2 for 14 h. The media were aspirated, and incubating CDTb with trypsin at a 1:5 (m/m) ratio for 45 min at 37 °C. The the cells were washed twice with 1× phosphate-buffered saline (PBS) before reaction was quenched with the addition of 0.1% PMSF and the preinsertion being fixed with 4% paraformaldehyde for 5 min and permeablized with state purified by size exclusion chromatography. To prevent aggregation of 0.1% Triton X-100 in 1× PBS for 5 min. The cells were then stained with this sample, LSR was mixed with the preinsertion state at a 2:1 molar ratio phalloidin-Alexa-488 for 20 min and washed twice with 1× PBS to remove before concentrating for downstream processing. excess dye. Cellular nuclei were stained with DAPI for 5 min and washed twice with 1× PBS. The coverslips were removed and mounted onto glass slides – – Cryo-EM Sample Preparation, Data Collection, and Processing. The CDTb LSR with Prolong Gold antifade reagent and were allowed to cure overnight. All CDTa complex was formed at a 1:2:2 molar ratio and applied to Quantifoil images were obtained on a Zeiss LSM 880 confocal microscope. R1.2/1.3 grids using a Vitrobot Mark IV at a concentration of 800 nM. Grids were plunged into liquid nitrogen-cooled ethane for vitrification. Data were Materials and Data Availability. Materials are available upon request. The collected on a Titan Krios operating at 300 keV with a Gatan K2 Summit data described in this publication are made available through the EM Data direct electron detector and Bioquantum energy filter set to a slit width of Bank (EMDB accession code 21016) and the Protein Data Bank (PDB acces- 20 eV (Washington University Center for Cellular Imaging; see SI Appendix, Table S2). All data were collected with a magnified pixel size of 1.13 Å/pixel, sion code 6V1S). − a total dose of 61.34 e /Å2, and a frame rate of 200 ms/frame for a total of 32 frames. These micrograph movies were corrected for beam-induced ACKNOWLEDGMENTS. The authors gratefully acknowledge James Fitzpatrick motion with MotionCorr2 with dose weighting applied (29). The defocus and Michael Rau (Washington University Center for Cellular Imaging) and values for all micrographs were estimated with Gctf, and a small subset of Scott Collier and Melissa Chambers (Vanderbilt University Cryo-EM Facility) for their help in cryo-EM sample preparation and data collection. We thank particles was initially picked and classified in Relion 3.0 to generate tem- members of the D.B.L. laboratory for their thoughtful feedback during plates for autopicking within Relion 3.0 (30). The picked particles were cleaned manuscript preparation. This work was supported by U.S. Department of over two rounds of two-dimensional classification and two rounds of three- Veterans Affairs Award BX002943, Public Health Service Grant AI095755 dimensional classification. The resulting map was corrected according to per- from the National Institutes of Health, and Vanderbilt University. M.J.S. was particle CTF values and Bayesian polishing before final refinement following a supported by the Gastroenterology training program (DK007673). All gold-standard refinement protocol in Relion 3.0 (31, 32). molecular graphics and model analyses was performed with UCSF Chimera which was developed by the Resource for Biocomputing, Visualization, and Model Construction and Refinement. The model of CDT was constructed by Informatics at University of California, San Francisco, with support from NIH first placing the models for the CDTb preinsertion state (PDB 6OKR) and CDTa P41-GM103311.

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