Complete structures of the YenTc holotoxin prepore and pore reveal the evolutionary basis for chitinase incorporation into ABC toxins

Domain: biology
Published: 2025-12-15
Editorial outcome: major_revision

Abstract
ABC toxins are toxin-translocating, pore-forming proteins found in a wide range of insecticidal bacteria and some mammalian pathogens. The Yersinia entomophaga toxin complex (YenTc) belongs to a distinct subclass of ABC toxins, defined by a divergent molecular architecture. Structural details that define their mechanism of action remain to be elucidated. Here we determine structures of the YenTc holotoxin assembly in both prepore and pore-forming configurations using cryo-EM in conjunction with Alphafold2-assisted structural modelling of flexible domains. We define the structural mechanism via which enzymatically-active chitinase subunits are incorporated, and show using phylogenetic analyses that this subclass-defining feature has evolved relatively recently. Our structures point to the existence of distinct conformational states in YenTc, which may distinguish it from other structurally-characterised ABC toxins, or represent states on a shared mechanistic trajectory. Thus, our findings enhance our understanding of the structural diversity that defines distinct ABC toxin subclasses.

Introduction
Yersinia entomophaga is an entomopathogenic bacterium that causes disease in susceptible insect hosts via targeted effects on midgut epithelial cells1–3. The major virulence factor associated with its pathogenicity is YenTc, a 2.4 MDa macromolecular assembly belonging to the ABC family of bacterial pore-forming toxins (also referred to as Tc toxins)1. ABC toxins are most commonly found in the genomes of insecticidal bacteria that are, for the most part, vectored by an nematode host4–6. Some mammalian pathogens also contain gene clusters that are either predicted or proven to encode for ABC toxins7,8. Y. entomophaga is unusual in that it is an insect pathogen that is able to both replicate and exert its toxic effects completely independently of any known nematode symbiont1,9.

YenTc and other ABC toxins share some functional similarities with the wider family of binary toxins, the latter being two-component toxins comprising an active component and a binding component10. The “active” component of YenTc (TcBC) is a heterodimeric complex of two proteins (YenB and YenC) which cooperatively fold to form a protective cocoon that harbours a cytotoxic enzyme11. The pathogenicity island encoding for YenTc includes two paralogs of YenC (YenC1, YenC2) which encode different predicted cytotoxic enzymes at their C-terminus; either of these can fold together with YenB to form the active component1. A third YenC paralog (YenC3) at a distant genomic location is also able to recombine with YenB to form a functional, active component12.

The “binding” component of YenTc (TcA) accounts for almost 90% of the complex by molecular mass. YenTc is a representative example of a distinct subtype of ABC toxins13–15 characterised by a TcA component that is comprised of four, separately encoded, unique polypeptide chains. Each polypeptide chain is present in five copies, yielding a 2.1 MDa complex13,16. Two of the four separately encoded polypeptides are functional chitinases17, while the remaining two polypeptides (YenA1 and YenA2) together form a predominantly α-helical structure that includes a pre-formed α-helical translocation channel, surrounded in its soluble state by an outer α-helical shell. Activation of YenTc occurs via an as yet undefined physiological trigger and leads to a large conformational change in the TcA subunit whereby the membrane-penetrating tip of the translocation channel becomes exposed13. Membrane engagement in vivo is likely to involve interaction with host-specific cell surface receptors that position the complex so that the tip is driven through an adjacent membrane bilayer13,18. In vitro studies suggest that YenTc engages with glycan motifs on the cell surface via the chitinase subunits13. The cell surface glycoprotein Vgr1 has been identified as a receptor for ABC toxins produced by Photorhabdus luminescens, but is not a receptor for YenTc19. Differences in the structure and architecture of the TcA component of YenTc, specifically the receptor-binding regions, offer the most likely explanation for these observed differences in specificity.

We previously reported structures of the separate TcA13 and TcBC components of YenTc11, but until now a structure of the combined, functional, holotoxin assembly has not been described. Here we report single particle cryo-EM structures of a native YenTc holotoxin in both the soluble prepore state and a lipid nanodisc-embedded configuration. The latter in particular represents a substantial improvement over previously reported structures, and therefore provides structural insights into the molecular transitions that accompany pore-formation. We identified structural differences between YenTc and other ABC toxins that may be representative of subtype-specific differences in structure or may alternatively represent different conformational states on a shared molecular trajectory. We were able to define the structural mechanism via which chitinase subunits (a defining feature of YenTc that distinguishes it from other ABC toxins) are incorporated into the holotoxin assembly. Phylogenetic analyses revealed that in a small, recently evolved subset of ABC toxins, chitinase-binding domains (CBDs) are inserted into the TcA α-helical shell at or near evolutionary hotspots which coincide with the location of putative receptor-binding domains (RBDs) in other ABC toxin subtypes. Unexpectedly, the CBDs share structural homology with bacterial adhesins, and the mechanism via which chitinases are incorporated shares similarities with the assembly mechanism seen in filamentous pili and fimbriae. Finally, we propose a refined classification system for bacterial ABC toxins and show that chitinase incorporation, as seen in the structure of YenTc, is a defining feature of a small subset of ABC toxins that has evolved relatively recently, suggesting this feature confers functional advantages resulting in enhanced fitness.

Results
Structures of native, functional YenTc holotoxin assemblies in the soluble, prepore configuration and reconstituted in lipid nanodiscs were determined by single particle analysis following imaging by cryo-EM. Notably, all of the datasets used in the course of this work were collected manually using a side-entry cryo-holder on a Tecnai F30 FEG-TEM (Field Emission Gun-Transmission Electron Microscope) fitted with a K2 Summit direct electron detector. Given that financial and geographical barriers often prohibit access to cutting-edge, automated microscopes our results serve as an important example highlighting the capabilities of previous generation instruments and thus contribute to the “democratisation” of cryo-EM20,21.

For the prepore structure, single particle images extracted from 616 micrographs were initially processed without any imposed symmetry, producing a structural map with an average resolution of 4.6 Å (Prepore map 1; Supplementary Fig. S1; Fig. 1A, B). Subsequent focused refinement of the TcA region with C5 symmetry imposed (Prepore map 2; Supplementary Fig. S1), and of the map density encompassing the TcBC subcomplex and the interfacing residues of TcA with no symmetry imposed (Prepore map 3; Supplementary Fig. S1), produced maps with resolutions of 4.0 Å and 5.7 Å for these regions, respectively.

To obtain the membrane-embedded pore form of the complex, we subjected complexes to mild chemical destabilisation by dialysis against guanidine hydrochloride at sub-denaturing (1.5 M) concentrations, prior to reconstitution into POPC lipid nanodiscs. Following size-exclusion chromatography, reconstituted pore-forms were identified in cryo-EM images, and single particle images extracted from a dataset of 1222 micrographs were processed with no symmetry imposed to produce a 3D reconstruction with a resolution of 6.4 Å (Pore map 1; Supplementary Fig. S1; Fig. 1C, D). Focused refinement on the TcA region, with imposed C5 symmetry, yielded a map with an average resolution of 5.9 Å (Pore map 2; Supplementary Fig. S2), substantially improving on the structure we reported previously at a resolution of 11 Å13 and allowing detailed interpretation of the molecular transitions associated with pore-formation.

Like other Tc toxins, and as suggested by our prior analysis of the individual components, the holotoxin structure of YenTc (Fig. 2A) features a central, pre-formed α-helical pore-forming domain that is surrounded by a predominantly α-helical outer shell that almost entirely encapsulates the pore. A funnel-shaped opening at the top of the pore-forming domain forms the interface with the TcBC component (TcB-binding domain). The asymmetric TcBC subcomplex breaks the otherwise fivefold symmetric structure. Model building was initiated by fitting our previously determined crystal structure of YenB-YenC2ΔCTD into the prepore holotoxin map. An initial model of the TcB-binding domain of TcA was extracted from our previously solved cryo-EM structure of the TcA component, and the two models were simultaneously refined against the map density.

Substantial remodelling of the TcA-TcBC interface was necessary, indicative of binding-induced structural changes in this part of the model. However, this was largely confined to the TcBC side of the interface (Fig. 2B–D and Supplementary Fig. S3C), with the TcA backbone remaining largely unchanged (Fig. 2E–G, RMSD = 3.33 Å across 750 residues). In the crystal structure of YenBC, the TcA-binding β-propeller domain adopts a distorted conformation (Fig. 2D), with two of the six blades closing the bottom of the cage. In the context of the holotoxin, the blades are repositioned, forming a less distorted, pseudo-symmetric structure (Fig. 2B), similar to what has been observed previously for the P. luminescens toxin complex 3 (PTC3)22. Thus, YenTc holotoxin formation involves an induced fit mechanism of binding and recognition between the TcBC and TcA components, where the TcBC component undergoes structural adaptation to match the interface presented by TcA (Supplementary Movie S1).

Given the similarity to the PTC3 system, we conclude that the structural mechanism via which assembly of the two toxin components into a functional holotoxin occurs is likely replicated in other members of the ABC toxin family. The rearrangements lead to the formation of a single, continuous fenestration linking the interior of the TcBC cocoon with the lumen of the TcA pore-forming domain. Residual, non-continuous map density, which could not be accounted for in the subsequent model building steps, can be seen within the lumen of the TcBC cocoon and likely corresponds to the unfolded toxin domain of YenC (Supplementary Fig. S4). This density extends into the lumen of the TcA pore-forming domain (Supplementary Fig. S4), indicating that the interface between the TcA and TcBC components is indeed open.

The transition of YenTc from the prepore to the pore conformation is characterised by substantial conformational changes that are almost exclusively confined to the TcA component. Whilst these changes are qualitatively similar to what was described in our previous manuscript that reported the structure of the TcA component reconstituted in liposomes13, the resolution of this nanodisc-embedded holotoxin assembly is substantially improved, facilitating a more confident and complete positioning of the individual structural domains and allowing us to derive insights into aspects of the prepore-to-pore transition.

Overall, the α-helical shell of YenTc undergoes an iris-like expansion (Fig. 3) that results in the loss of all interchain contacts between YenA1 protomers, meaning the only points of contact remaining within the α-helical shell are between adjacent YenA2 protomers. Shell expansion accommodates a vertical displacement of the pore-forming domain and the attached YenBC cage. The pore-forming domain moves relative to the surrounding YenA shell by a distance of ~170 Å, resulting in the YenBC cage being almost completely enclosed within the expanded shell (Fig. 3A). The iris-like opening of the YenA shell is accompanied by a compound hinge-bending motion about two points in the structure, located on either side of the YenA1-YenA2 interface (the interface itself remains unchanged). The first hinge is located within the YenA2 region of the shell, and the bending motion results in an upward rotation of the lower part of the shell, the attached neuraminidase-like domain and CBD-B by ~15° (Fig. 3D). Movement about the first hinge point results in separation of the neuraminidase-like domains, making room for the emergence of the pore-forming domain from the base of the shell. The second hinge is within the YenA1 region of the shell, again close to the YenA1-YenA2 interface, and leads to a more subtle movement of the rest of the YenA1 shell and attached CBD-A. As a result, the opening of the shell is slightly more pronounced than what would occur if it rotated purely as a rigid body.

To our knowledge, the PTC3 toxin is the only other ABC toxin that has been structurally characterised in its pore-forming state. When comparing the YenTc pore with PTC3, the most striking difference is the degree to which the pore-forming domain is ejected out of the surrounding α-helical shell. Notably, only the TcB-binding domain and the attached TcBC cage remains inside the expanded shell of YenTc. This contrasts with PTC3, where the ejection of the pore-forming domain from the shell appears to be less extensive (Supplementary Fig. S5). In quantitative measures, the pore-forming domain moves relative to the surrounding shell by an additional 40 Å in YenTc compared to PTC3. The transition from prepore to pore in ABC toxins generally is accompanied by folding of an extended linker in the prepore state into a more compact α-helix in the pore form; this structural transition either drives or is a consequence of pore-formation.

Interestingly, the folded linker domain in YenTc (Supplementary Fig. S5) is three turns longer than the equivalent helix in PTC3 (Supplementary Fig. S5C). Our observation of a more extensively folded linker helix in YenTc is likely a consequence of the more extensive pore ejection.

In the YenTc pore, the inner face of the linker helix packs against the TcB-binding domain, which represents an additional point of difference to the structural configuration seen in PTC3. To explore this further, we performed an in-depth analysis of the respective linker interfaces in PTC3 and YenTc (see Text S1) and concluded that poor sequence conservation in a structural element we termed the 'collar loop' may explain the differences in linker positioning. We also examined the corresponding sequences in other ABC toxins that have been structurally characterised in the prepore state23 and found that this collar loop motif has a well-conserved sequence in all toxins we examined, with YenTc being the one exception (Supplementary Fig. S6 and Text S1). Notably, YenTc is the only example among this group that has a split TcA subunit architecture. Our analysis, therefore, suggests that the YenTc pore form could be representative of a distinct structural conformation that is potentially also adopted by other ABC toxins belonging to subtypes that have a split TcA subunit.

While our previous structural studies of YenTc identified the locations of the Chi1 and Chi2 domains within the TcA component of YenTc, poor local map resolution meant we had been unable to resolve the Chi2-YenA1 and Chi1-YenA2 interfaces13. Thus, the structural mechanism via which chitinases are incorporated into YenTc (and indeed, into any ABC toxin) has remained unclear. The improved maps reported here allow unequivocal identification of two previously unresolved CBDs inserted into the TcA shell (Fig. 4A). CBD-A (YenA1 residues 311–472) forms the major binding interface with the Chi2 subunit of TcA and could be modelled based on the map density alone. CBD-B (YenA2 residues 400–520) forms the major binding interface with the Chi1 subunit, but due to substantial conformational dynamics, required a hybrid modelling approach (detailed below).

CBD-A adopts a classical β-sandwich fold and a DALI search24 for structurally related proteins identified the adhesin domain of the Escherichia coli fimbriae protein CfaE (RMSD = 2.9 Å) as its nearest structural homologue (Supplementary Fig. 7B). Curiously, CfaE also forms heterodimeric interfaces involving β-donor and β-acceptor interactions that are remarkably similar to the interaction seen between CBD-A and Chi2NTD. In the context of CfaE, the interaction seeds the formation of a filament of polymeric pilin proteins linked via a daisy chain of β-donor/β-acceptor interactions25. The nature of the β-strand interface between Chi2 and CBD-A differs slightly from that seen in CfaE, however, in that CBD-A has adapted a region that acts as a stabilising loop in the CfaE structure to act as the CBD-A β-donor (Supplementary Fig. 7C). This motif in turn recruits the N-terminal β-acceptor from Chi2NTD, and an extension of the Chi2NTD beyond the β-acceptor appears to act as a stabilising loop (Supplementary Fig. S7C). Thus, the mechanism of polymeric pilin assembly appears to have been co-opted here to facilitate recruitment of Chi2 into a stable complex with YenTc.

A 3D variability analysis using our cryo-EM data confirmed that substantial conformational dynamics (Supplementary Movie 2) are the most likely contributing factor to the poor local map resolution in the region of the Chi1:CBD-B interface, prohibiting high-resolution structure determination. To model this interface, we therefore employed a hybrid model-building approach using AlphaFold2 multimer (AF2m), cross-linking mass spectrometry (XL-MS), MD simulations and structural data derived from our previously determined crystal structure of the Chi1 subunit17. The AF2m-predicted structure of CBD-B (Supplementary Fig. S8A) showed a reasonable fit to the map density in the CBD-B region (Fig. 4D), and the predicted Chi1 structure was unsurprisingly consistent with the crystal structure. The absolute positioning of the attached Chi1 subunit was somewhat inconsistent with the residual map density attributed to Chi1 in the full complex, which we again expect is due to the extreme flexibility evident in this region. To validate the AF2m predicted interface, we performed XL-MS analysis on the holotoxin assembly. Three crosslinks between Chi1 and CBD-B (Chi1 E399-YenA2 K447, Chi1 K448-YenA2 K449, Chi1 E527-YenA2 K449) were identified (Supplementary Fig. S8B, C), two of which were satisfied in the AF2m structural model (Fig. S8B). To account for flexibility, as a final validation step, we performed triplicate, 1 µs MD simulations focused on this region of the holotoxin structure, and found that in addition to the predicted interface remaining stable throughout all simulations, all three contacts identified in our XL-MS analysis were satisfied throughout the simulations (Supplementary Fig. S8C). Thus, we concluded that our hybrid model provides a robust representation of the Chi1:CBD-B interface.

Chi1 lacks the 90 amino acid N-terminal domain found in Chi2 and is instead anchored to CBD-B via a single, unstructured strand located at its C-terminus (Chi1 CTS) that is not resolved in the crystal structure17. Chi1 CTS integrates with an exposed hydrophobic cleft formed by CBD-B (Fig. 4C), and while the involvement of a hydrophobic cleft is somewhat similar to the interaction seen between CBD-A and Chi2NTD, the Chi1 CTS is predicted to adopt an extended strand topology in binding along the cleft, rather than the β-strand topology adopted by Chi2NTD. Notably, the Streptococcal factor H-binding protein (Fhb) and adhesin (SadP) are identified by DALI as structural homologues of CBD-B (Supplementary Fig. S9). Thus, CBD-A and CBD-B both appear to have features in common with bacterial adhesins or adhesin-like domains, leading us to speculate that this may represent a preferred structural fold for the anchoring of chitinases within the α-helical shell of ABC toxins.

While CBD-B shares structural homology with the adhesin proteins noted above, the closest structural homologues of CBD-B detected by DALI are putative receptor binding domains of other TcA proteins, in particular the RBD-A, RBD-B, and RBD-C domains of the TcA component from the P. luminescens PTC3 toxin, which are conserved to varying extents in other ABC toxins23,26. CBD-B shares a strikingly similar structural fold with all three RBDs identified in the PTC3 structure (RMSDs range from 2.6 to 3.4 Å), despite having low sequence identity (9–20%) (Supplementary Fig. S9). This low degree of sequence conservation provides a plausible explanation for why CBD-B (but apparently not the RBDs of other TcA proteins) facilitates incorporation of chitinase subunits into a holotoxin assembly.

Aside from the extent to which their sequences have diverged, a further difference exists with respect to the structural positioning of CBD-B relative to the RBDs found in other TcA proteins. When the prepore structures of YenTc and PTC3 are overlaid, CBD-B is rotated downwards through an angle of ~107° relative to the position occupied by RBD-B in PTC3 (Fig. 4D). Interestingly, independent experiments conducted on PTC3 during the course of our studies27 provided evidence for the existence of structural intermediates between the prepore and pore conformations, one of which repositions the RBD-B/C region into a downward-facing orientation that more closely mirrors the positioning of CBD-B and the bound Chi1 subunit in YenTc (Supplementary Fig. S10). This led us to explore whether it was possible for the reverse motion to occur in YenTc. Repositioning of CBD-B, such that it mirrors the upward conformation seen in PTC3, was not possible without additional and substantial structural rearrangement of the rest of the complex. Moving CBD-B as a rigid body introduces steric clashes between the attached Chi1 subunit and the Chi2 subunit associated with the neighbouring protomer. Thus, binding of the Chi1 and Chi2 subunits appear to sterically preclude CBD-B from adopting a similar prepore configuration to the RBD domains seen in other ABC toxins that do not incorporate chitinases.

We also noted that the CBD-B domain of YenA2 not only shares structural homology with the putative RBDs of other TcA proteins, but that CBD-B is inserted into the same structural loop of the conserved neuraminidase-like domain. Thus, it appears likely that CBD-B may have been adapted from an RBD (although the converse may also be true, i.e. that other RBDs are adapted from CBD-B). Alternatively, this specific loop in the neuraminidase-like domain may represent an evolutionary hotspot, within which (at least) two different domains implicated in receptor recognition have been inserted via separate evolutionary events.

We investigated whether a similar, evolutionary hotspot exists within the α-helical shell of TcA, where CBD-A is inserted into YenTc and RBD-A is inserted into PTC3. Surprisingly, however, we found that RBD-A and CBD-A are not inserted into the same location, with CBD-A inserted ~200 residues closer to the N-terminus of YenA1 relative to the corresponding insertion site of RBD-A (Supplementary Fig. S7A). Attempts to superimpose the two domains also revealed little structural similarity between CBD-A and RBD-A, further supporting the conclusion that incorporation of these domains likely occurred via independent evolutionary events. Additionally, YenTc appear to have retained a small domain with no regular secondary structure in place of the canonical RBD-A fold. This 'pseudo RBD-A' domain is not found in the shell of other, structurally characterised ABC toxins that lack an RBD-A (for example, the P. luminescens TcdA4 protein). This suggests that, from an evolutionary perspective, toxins containing the RBD-A domain likely emerged prior to those containing the CBD-A domain (typified by YenTc), and that the RBD-A domain became the pseudo RBD-A domain that remains in YenTc, either soon before or subsequent to CBD-A being acquired.

To provide an enhanced context for understanding the evolutionary events that led to insertion of the CBD-A and CBD-B domains, facilitating the direct structural association of chitinases with ABC toxins, we constructed phylogenetic trees based on a curated database of ABC toxin loci assembled from published genomes. A detailed description of how this database was assembled is provided (see “Methods” and Text S2). The dataset was initially curated so that only complete ABC toxin loci were included (a toxin locus was considered complete if it contained a TcA component predicted to include a TcB-binding domain, and at least one copy of a TcB and a TcC component). To facilitate unambiguous association between components, a toxin locus was only included in the analysis if there were no other identifiable toxin components within the genome from which it was derived, or if unequivocal association between components could be inferred from other forms of evidence (e.g. from structural studies, as is the case with several Photorhabdus and Xenorhabdus toxins).

When the filtered database of toxin loci was subject to phylogenetic analyses, four clearly separated and well-defined clusters emerged when trees were constructed based on the alignment of TcA subunits (Fig. 5). Two of these clusters represent ABC toxins that have a split TcA gene. In a previous study, we defined these split TcA toxins as Type 2 toxins13,14 and it was evident in our analysis here that these separate further into two distinct phylogenetic clades. One clade includes toxins like YenTc, that incorporate chitinases into their TcA component while the other has a split TcA gene but no predicted chitinase genes within 100 bp of the toxin locus. For the sake of consistency with our previously defined nomenclature, we refer to these two clades hereafter as Type 2A (split TcA with chitinases) and Type 2B (split TcA without chitinases).

We searched the entire sequence database for evidence of CBD conservation within the various TcA sequences. Using hidden Markov model sequence profiles, we were unable to find sequences matching the profiles constructed for CBD-A and CBD-B in any other TcA proteins, outside the limited number of Type 2A toxins present in our dataset. This was somewhat unsurprising given that Type 2A toxins were also the only toxins for which chitinases appear to be co-localised with other toxin components, and supports their role as bona fide CBDs. We subsequently searched the entire UniProtKB database for CBD-A and CBD-B domains and detected these in 23 genomes, predominantly from the Xenorhabdus sp., but also Photorhabdus asymbiotica, Photorhabdus laumondii, and Yersinia nurmii (Supplementary Fig. S11). The additional sequences detected in this way are all derived from genomes that include multiple TcA components and were therefore not included in the phylogenetic analysis. However, only TcA components that included hits for CBD-A or CBD-B were flanked by putative chitinases. Thus, we conclude that either a CBD-A or a CBD-B domain is a requirement for chitinase incorporation.

The phylogenetic analysis undertaken here additionally revealed that at least two separate clades of single TcA toxins exist. The smaller, more recently evolved clade includes the P. luminescens PTC3 as well as toxins incorporating the P. luminescens TcdA4 subunit, Xenorhabdus nematophila XptA1 subunit, and Morganella morganii TcdA4 subunit23. The larger clade appears to be the most phylogenetically ancient clade, and to date no toxins belonging to this clade have been structurally characterised.

Using the same hidden Markov model sequence profile analysis invoked to search for CBD-A and CBD-B, we built profiles representing the RBDs previously identified within solved structures23 or defined within InterPro (see Supplementary Text 2) and found that RBDs were exclusively conserved (albeit to varying extents) within TcA sequences belonging to the more recently evolved of the two, single TcA clades identified above. RBD-D was the most strongly conserved, found in 77.8% of the sequences belonging to this clade, with RBD-A, -B and -C less well conserved. In a previous analysis conducted by Song et al. it was independently found that the prevalence of RBD-D domains in Type 1 ABC toxins was 83.4%15. We therefore conclude that Type 1 ABC toxins are characterised by the presence of a single TcA component, with a high prevalence of RBD-D domains and variable rates of incorporation of PTC3-like (and potentially other) RBDs.

Importantly, we could not find any evidence supporting the conservation of the currently defined RBDs or CBDs in the more ancient clade of single TcA toxins. Sequences from this clade are vastly under-represented in the independent analysis conducted by Song et al15. Thus, we conclude that this subset of TcA sequences is indicative of a previously undefined clade of ABC toxins, which we defined as Type 3 ABC toxins. In addition to the absence of any CBDs or RBDs, we also noted that members of this clade were characterised by a fused TcBC protein, rather than having separate TcB and TcC components as is typical of the other clades. We constructed additional phylogenetic trees based on the TcB and TcC components (for the purpose of this analysis, TcBC fusions were split into two sequences and classified separately) and found that broadly, the clustering of the TcB and TcC sequences mirrored that seen for the TcA sequences (Supplementary Fig. S12).

Our phylogenetic analyses therefore suggest that – as typified by the Type 3 architecture — ABC toxins were originally two component toxins comprised of TcA and TcBC components. Given the high bootstrap values on the branches between Type 1, 2 and 3 toxins, and on the branch between Type 2A and 2B toxins (Fig. 5), we can be confident that Type 1 and 2 toxins emerged subsequently, following the fission of TcBC into two separate components, and that a second fission event in the TcA component produced Type 2A and Type 2B toxins. Our combined structural and phylogenetic analyses further suggest a plausible evolutionary scenario giving rise to the distribution of RBDs and CBDs and delineation of the more recently evolved Type 1 and 2 clades. Because the P. luminescens PTC3 (Type 1) RBD-B and YenTc (Type 2A) CBD-B domains are similar in their structure and insertion point (Fig. 4D), we hypothesise they are orthologous domains derived from a common RBD-like β-sandwich domain acquired by the ancestors of Type 1 and 2 toxins after splitting from Type 3 toxins. Following another split within the Type 2 toxins, the 2A clade adapted these domains for recruitment of chitinases (which we infer as a recent evolutionary event from the phylogenetic tree), while the 2B clade appears to have lost these domains. Type 2A toxins likely acquired CBD-A in a separate evolutionary event given it has no structural similarity—nor does it share an insertion point—with any RBDs of known Type 1 toxin structures (Supplementary Fig. S7A). The identification of a pseudo RBD-A domain in YenTc and other Type 2A toxins (Fig. 5) indicates that this domain likely descended from RBD-A of Type 1 toxins and was lost via genetic drift, and further supports the conclusion that Type 2 toxins evolved most recently.

Methods
Bacterial strains used
The Y. entomophaga toxin complex (YenTc) was purified from wild-type Y. entomophaga (strain MH96). E. coli BL21 DE3 was used for expression of membrane scaffold proteins (MSP1D1) for preparing lipid nanodiscs.

Expression and purification of YenTc
Full-length YenTc was expressed and purified as described previously16,33. In brief, Y. entomophaga was cultured in antibiotic-free LB medium at 25°C overnight, shaking at 180 rpm. Cultures were incubated at 25°C for 1 h without shaking to allow for YenTc secretion, then harvested by centrifugation at 4800 × g to pellet the cells, and the media supernatant collected. Ammonium sulphate was added to the supernatants to a final concentration of 70% (w/v) and the precipitated protein collected by centrifugation at 13,000 × g, following which the remaining supernatant was discarded. Precipitated protein was resuspended in TBS buffer (50 mM Tris, 150 mM NaCl, pH 7.6), filtered, and purified further by Superose 6 (Cytiva) size-exclusion chromatography. Fractions corresponding to YenTc were pooled and concentrated for further experiments.

MSP1D1 nanodisc scaffolding protein expression and purification
A plasmid vector containing the scaffolding protein MSP1D1 gene (provided by Dr Carus Lau) was transformed into E. coli BL21 DE3 for expression. Expressed protein was purified by gravity flow immobilised metal affinity chromatography as described34 and dialysed into TBS buffer. Nanodiscs (NDs) were prepared as described previously34.

Formation and reconstitution of pore-form YenTc in nanodiscs
To induce pore formation, YenTc was first subjected to dialysis against buffer containing guanidine hydrochloride (GnHCl) and Tween-20 (50 mM Tris, 150 mM NaCl, 1.5 M GnHCl, 0.05% Tween-20, pH 7.6). A second dialysis step was then performed using buffer without GnHCl. This pre-solubilised, pore-form YenTc sample was then mixed with POPC lipids solubilised in buffer containing sodium cholate (50 mM Tris, 150 mM NaCl, 23.2 mM sodium cholate, pH 7.6) and incubated for 30–45 min at 4°C. Subsequently, MSP1D1 was added, and the sample was incubated for a further 1 h at 4°C, achieving a final cholate concentration of 16–18 mM and molar ratios of 1:16:440 for YenTc:MSP1D1:POPC. Detergent removal was achieved in one of two ways—either through overnight incubation with Biobeads-SM2 (Biorad), or through dialysis against detergent-free TBS buffer—following which the sample was subjected to Superose 6 (Cytiva) size-exclusion chromatography.

Cryo-EM grid preparation and data collection
R2/2 holey carbon grids (Quantifoil) or lacey carbon grids with graphene oxide support film (ProSciTech) were washed using ethyl-acetate prior to glow discharge. Vitrification of YenTc prepore and pore-form samples was performed using a Vitrobot Mark IV (ThermoFisher Scientific) operating at 4°C and 100% humidity. 4 μL of each sample (0.2–1.1 mg/mL in TBS) was applied onto a grid and allowed to absorb for 3–10 s. The grid was then blotted for 3–4.5 s before plunge freezing in liquid ethane in preparation for cryo-EM. Data collection for both YenTc prepore and pore-form samples was performed on a Tecnai F30 FEG-TEM (ThermoFisher Scientific) operating at 300 kV and fitted with a K2 Summit direct electron detector (Gatan). A nominal magnification of 23,000×, corresponding to a pixel size of 1.72 Å at the specimen level, was used for data collection. Datasets were collected using SerialEM software with dose fractionation in electron counting mode, with 40 subframes collected per image and a total dose of 45–49.2 e/Å2 (dose rate 8–9 e/pix/s). The defocus range used was from 1.8 to 3 μm.

Single-particle processing of YenTc prepore and pore images
For the YenTc prepore, a total of 616 movies from three data collection sessions were merged and processed using cryoSPARC (version 3.2.1)35. Patch-motion correction and patch CTF estimation were performed, and 137,309 particles were picked. Multiple rounds of 2D classification filtered the dataset to 72,265 particles, and these particles were used to generate an ab initio model. The best ab initio model (60,315 particles) was subjected to homogenous refinement using C1 and C5 symmetries, resulting in maps with average, estimated resolutions (FSC=0.143) at 4.6 Å and 4.0 Å, respectively. Focused refinement of the BC domain was performed, yielding a map at 5.7 Å. The final data collection and modelling parameters are shown in Table S1 and the processing strategy is summarised in Supplementary Fig. S1.

For the YenTc pore, 1222 movies collected across multiple sessions were patch-motion corrected with dose-weighted averaging using cryoSPARC (version 3.2.1)35. CTF parameters were estimated with patch CTF, and 116,252 particles were picked using the template picker. Following particle curation and 2D classification, 97,198 particles remained. To overcome issues with preferred particle orientation, the particles were re-distributed in six new superclasses using 2D class rebalancing, which reduced the particle dataset to a total of 40,973 particles. These were used to generate two ab initio models, one of which exhibited clear density for the pore domain of YenA. This ab initio model was used as input for homogeneous, non-uniform and heterogeneous refinements. The final models were refined using C5 and C1 symmetry, producing final maps with average, estimated resolutions (FSC=0.143) of 5.9 Å and 6.4 Å, respectively. The final data collection and modelling parameters are shown in Table S1, and the processing strategy is summarised in Supplementary Fig. S2.

Model building and refinement of YenTc prepore and pore structures
Model building into the YenTc prepore and pore maps was initiated using previously published models for the TcA (6OGD) and TcBC (4IGL) subunits. Newly-resolved structural domains that were missing from these models were built using COOT 0.8.9.336. AlphaFold2 (AF2) multimer37 was used to generate an initial structure of the CBD-B-Chi1 interface, using residues 400–520 of YenA2 and the complete sequence of Chi1 as inputs. The source coordinates are summarised graphically in Supplementary Fig. S13. The entire structure was then subject to iterative refinement cycles using ISOLDE 1.6.038 and Phenix v1.21.520739. For the YenTc pore form, within most domains there were few structural changes between the prepore and pore form, and these were able to be built initially by splitting the prepore structure into domains and transferring the corresponding coordinates one domain at a time from the prepore model into the pore map density. For the remaining regions, the Cα backbone was traced in COOT, side chains added, and then the entire assembly was refined using ISOLDE and Phenix. Side chains were used to assist model refinement, but were stubbed in the final deposited model as the resolution of this map was considered insufficient to reliably infer side chain conformations.

Protein cross-linking, digestion mass spectrometry and peptide identification
YenTc used for crosslinking experiments was prepared in essentially the same way as for structural studies, with the exception that it was necessary to purify in HEPES-buffered saline. For each crosslinking experiment, ~50 µg of the complex was used, and experiments were performed essentially as described elsewhere40. Azido-benzoic-acid-succinimide (ABAS) crosslinking experiments were performed in the same manner as for the sulfosuccinimidyl 4,4’-azipentanoate (sulfo-SDA) crosslinker, with the exception that 254 nm UV was used for ABAS.

Liquid chromatography and mass analysis parameters were essentially as described elsewhere41 with the following exceptions: (1) Mass analyses were performed using an Eclipse Tribrid mass spectrometer (ThermoFisher Scientific); (2) For sulfo-SDA, ABAS and DMTMM, following each full-scan MS1 at 60,000 resolution at 200 m/z (300–1800 m/z, AGC=4×105, 50 ms max injection time), precursor ions were selected for sequential HCD-EThcD-MS2 acquisitions in a data-dependent manner (HCD-MS2: R=15,000, 30 NCE; EThcD-MS2: R=15,000, calibrated charged dependent ETD parameters enabled, supplemental HCD at 30 NCE; both: 22 ms injection time, 1.4 m/z isolation window, 5×104 intensity threshold, charge states +4–10, dynamic exclusion of 20 s).

DSSO and DHSO cross-linked peptides were identified using the XlinkX 2.042 nodes as implemented in Proteome Discover v2.3 (ThermoFisher Scientific). Sulfo-SDA, ABAS and DMTMM datasets were processed with MS Studio (v2.4.0.3545)43. Generally, the following key parameters were used: peptide mass between 300 and 10,000 Da, minimum peptide length of 5–6 residues, precursor mass tolerance ±10 ppm, product-ion mass tolerance of ±20 ppm for Orbitrap data and ±0.5 Da for ion trap data. Allowable variable modification = oxidation (M), allowable static modification = carbamidomethyl (C), enzyme specificity of Trypsin with up to two (DSSO, DHSO) or four (Sulfo-SDA, ABAS, DMTMM) missed cleavages (excluding the site of cross-linking), and FDR control at 1%. The search database used consisted of YenTc proteins. The S20D4 default search setting was initially used for DSSO and DHSO datasets but filtered to S80D20 on manual spectral verification. Cross-linked spectral matches from MS Studio were also manually visually assessed. All mass spectrometry data and XL-MS search results have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository44 with dataset identifier PXD068275.

Molecular dynamics structure preparation and simulations
Simulations to validate both the AF2m-predicted interface and the identified Cα-Cα distances from crosslinking mass spectrometry experiments between CBD-B and Chi1 were performed using the GROMACS 202245 simulation package in conjunction with the united-atom GROMOS 54a7 forcefield46. To prepare the system for simulations, the CBD-B:Chi1 structure was placed in a triclinic box with dimensions 13 nm × 9 nm × 9 nm and solvated with 33,385 water molecules. A single point charge47 model for water was used. Na+ and Cl- ions were added to neutralise the system charge and reach a final ionic strength of 150 mM. The final system, containing 107,112 atoms, was energy minimised using a steepest descent algorithm with a tolerance of 250 kJ.mol–1.

The system was equilibrated for 5 ns in an NPT ensemble using a 2 fs timestep and position restraints with a force constant of 1000 kJ.mol–1.nm-2 applied to all atoms. Subsequently, a series of 5 ns NPT simulations were performed with decreasing positions restraints of 500 kJ.mol–1.nm-2, 250 kJ.mol–1.nm–2, 100 kJ.mol–1.nm–2 and 20 kJ.mol–1.nm–2.

Production runs were carried out in triplicate for 1 µs using a 4 fs timestep. Non-bonded interactions had a cutoff of 1.4 nm with the neighbour list updated every 5 steps and were modelled using the Verlet scheme. Electrostatic interactions beyond the cutoff were modelled using the reaction-field method48. All system components were temperature coupled using a Berendsen thermostat49 with a temperature of 300 K and a time constant of 0.1 ps. All components used a Berendsen barostat with isotropic pressure coupling, with a compressibility of 4.5 × 10–5 bar–1 and a time constant of 1 ps. The periodic boundary conditions were applied in the x, y, and z dimensions.

Analysis of molecular dynamics data
Cluster analysis was performed on backbone atoms using the linkage method with a 0.15 nm cutoff in GROMACS 2022 on both the individual simulated replicates and a concatenated trajectory containing all three replicates, to ensure the conformations of the protein were independent from the starting structure (Supplementary Fig. S14).

From each simulation, the complete trajectory (1250 frames) was used to track the distances between residues identified in XL-MS experiments between CBD-B and Chi1. MDAnalysis50,51 was used to calculate distances between crosslinked atoms over the course of the simulation. Distance over time plots were used to ensure distances had converged for the duration of the production simulations (Supplementary Fig. S14). For all residue pairs identified in the crosslinks, the Cα-Cα and reaction centre distances between the residues were tracked. For the DSSO crosslink identified between K447 of CBD-B and K448 of Chi1, the reaction centres were defined as the NZ atoms of each side chain. For the DMTMM crosslinks identified between K449 of CBD-B and E527 and E399 of Chi1, the reaction centres were defined as the NZ atom of lysine and the OE1 atom of glutamic acid.

Bioinformatic analysis and phylogenetic tree building
Refer to Supplementary Information.

Structure visualisation and analysis
Structural data were visualised using UCSF ChimeraX-1.3. Multiple sequence alignments generated for the purpose of structural comparisons (i.e. excluding the phylogenetic analysis detailed above) were produced using the Molecular Evolutionary Genetics Analysis software52 using ClustalW with default parameters53. Structural homologues of the CBD-A, CBD-B and Chi2NTD were identified using DALI24 and TM-align54. Interface analyses were performed using PDBePISA to identify residues involved in electrostatic interactions55.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
All data are available in the main text or the supplementary materials. Structures presented in this study have been deposited in the Electron Microscopy Data Bank and Protein Data Bank under the following accession codes: YenTc holotoxin prepore EMDB-45190 [https://www.ebi.ac.uk/emdb/EMD-45190], PDB-9C4K; YenTcA prepore EMDB-45405 [https://www.ebi.ac.uk/emdb/EMD-45405]; YenTcBC prepore EMDB-45409 [https://www.ebi.ac.uk/emdb/EMD-45409]; YenTc holotoxin pore EMDB-45422 [https://www.ebi.ac.uk/emdb/EMD-45422], PDB-9CBC, YenTc pore EMDB-45423 [https://www.ebi.ac.uk/emdb/EMD-45423]. The mass spectrometry data used in this study are available in the ProteomeExchange via PRIDE under accession code PXD068275. Molecular dynamics trajectories and data have been deposited to Zenodo [https://doi.org/10.5281/zenodo.17438278]. The following PDB structures were referred to in the text: 4O9Y; 6YRO; 5BOA; 6HBO; 5UD7; 6DFQ; 4IGL; 6OGD; 6RW8; 6RWA; 6RW9; 6RWB.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Review of \"Complete structures of the YenTc holotoxin prepore and pore reveal the evolutionary basis for chitinase incorporation into ABC toxins\"\nThis manuscript presents a detailed structural analysis of the YenTc holotoxin from Yersinia entomophaga, using single-particle cryo-EM and AlphaFold2-assisted modeling to determine its architecture in both prepore and pore-forming states. The authors provide a mechanistic explanation for chitinase incorporation within the toxin structure, supported by phylogenetic analyses that highlight its recent evolutionary emergence. The study significantly advances the understanding of ABC toxin diversity, structure, and function, providing key insights into their mode of action and evolutionary origins. However, additional validation and clarification are needed to justify publication in Nature Communications. Addressing the concerns regarding resolution, data collection, refinement protocols, and model validation will significantly strengthen the manuscript.\nMajor Strengths\n1. Structural Insights: The determination of both prepore and pore configurations provides a comprehensive view of YenTc's conformational transitions, enhancing our understanding of ABC toxin assembly and activation.\n2. Mechanistic Elucidation: The identification of chitinase-binding domains (CBD-A and CBD-B) offers novel insights into protein-protein interactions within ABC toxins. The structural basis of chitinase incorporation into YenTc represents the first structural example of a novel protein/subunit recruitment mechanism, adding significant novelty beyond their previous work.\n3. Evolutionary Perspective: The phylogenetic analysis strengthens the manuscript by positioning YenTc within the broader ABC toxin family and revealing the evolutionary significance of chitinase incorporation.\nMajor revisions:\n1. Resolution limitations: Some structural elements, particularly the CBDA-Chi2 and CBDB-Chi1 interfaces, appear less resolved. The authors attempt to address this using hybrid modeling and combine it with MD and XL-MS to validate their structure experimentally. However, additional information should be provided to evaluate the structural quality and assess whether their claims are experimentally supported.\nAccording to nature's reporting standards, official validation reports from the wwPDB are required for peer review of macromolecular structures (https://www.nature.com/nature-portfolio/editorial-policies/reporting-standards). Please provide these reports for the structures deposited in the PDB (YenTc holotoxin prepore and pore structures).\nAlso, a figure showing the cryo-EM map of CBDA-Chi2 and CBDB-Chi1 with their corresponding atomic models should be included in the main manuscript.\n2. Structural quality concerns: Figure 2D shows that the secondary structure of the two blades undergoing major conformational changes is lost. In PTC3, each blade contains 3-4 beta-strands, suggesting that the refinement protocol used for YenTc may need revision, including correct secondary structure restraints if necessary.\n3. Data collection concerns: The study collected only 616 and 1222 movies for the prepore and pore conformations, respectively. Could additional data collection (e.g., 10x more data) improve resolution and model quality? The authors should discuss this limitation and justify their data collection strategy.", "reviewer_2": "In review of the manuscript, \"Complete structures of the YenTc holotoxin prepore and pore reveal the evolutionary basis for chitinase incorporation into ABC toxins.\"\nThis study presents two low-resolution cryo-EM structures of YenTc, one in its holotoxin prepore complex form and the other in its putative ‘pore’ conformation. The authors used AlphaFold to model a previously unresolvable segment, the chitinase-binding domain (CBD), and used bioinformatics to further characterise the evolutionary history of these proteins.\nABC toxins of Yersinia represent a fascinating class of functional proteins. However, we find that this manuscript offers only an incremental advance over previously reported structures of YenTc, raising concerns about its true novelty. While the resolution is an improvement over prior models, it remains relatively modest and still necessitates the use of AlphaFold to complete the structural model (previous publications show the pore and prepore structures of TcA (Piper et al. (2019)), the complex of TcBC (Busby et al. (2013)), as well as 2D classes and models of the full YenTc holotoxin (Landsberg et al. (2014)). Due to the limited data presented, the manuscript tends to over-discuss minor details, which impacts the overall readability and engagement of the text. This is a strong enough feature to make the results section read much like a discussion section in places.\nThe informatics component, and statements regarding domain insertion and interaction (e.g. page 10 line 20 “may provide the first structural example of a novel protein/subunit recruitment mechanism”) are essentially true of ANY system and are descriptive of natural variation rather than any especially interesting mechanism. A similar scenario exists in the over-documentation of the linker helix extension – there is not enough importance in essentially just discussing minor variations.\nMinor points:\nAbstract – “we determine structures …using cryo-EM and Alphafold2-assisted structural modelling”; determination (experimental) and modelling (in silico) should not be conflated/linked in this manner and require separating or rewording\nLine 67: “pentameric structure” – Describing TcA as pentameric is incorrect as its is not 5 polypeptides, but a c5 symmetric icosmeric complex made of a 4-sub unit protamer.\nLine 74: “juxtaposed” – The usage of this word is incorrect as its used to highlight contrast between two adjacent objects.\nLine 91: Chitinase binding domain (CBD) is a poor choice of acronym, as it is highly used, including ‘carbohydrate binding domain’, and the very similar ‘chitin binding domain’.\nFigure 1 AC: should the cream coloured chitinase be yellow as its part of the yellow protamer?\nFigure 2A-E: there is a substantial conformational change in the protein, however there is no evidence of the density that was used to support this. Is this region an Alphafold model or was it morphed from the closed structure? As the local resolution appears to be between 5-7 Å, it would be good to have figures of the model and associated density in the supplementary.\nLine 271-273 and line 323-325: the first states the “poor local map resolution had until now prevented us from building structures”, suggesting that the resolution now allows building of the model. However the second section states “The resolution of our maps in the region of the Chi1 region is more modest…while this prevented de novo building, a hybrid model building approach [with AlphaFold2] proved successful”. These statements are contradictory and the first perhaps suggests that the data are better than they are and so requires toning down\nFigure 2 legend should read “TcB and TcC are in pink”\nFigure 4 line 285: RBD-like domains – should be referred to as CBD domains for continuity.", "reviewer_3": "This work focuses on the structural biology of YenTc, a representative element of a subclass of the ABC toxins. The mechanism of action of these toxins has not yet been fully clarified. While previous work from the authors yielded the structure of the “binding” and the “active” subunits of this macromolecular complex, the present work provides the missing structural details, including models for the prepore and the pore forming structures.These are resolved by combining cryoEM experiments with bioinformatic-based structural modeling (via Alphafold-2).\nThe difference between the YenTc’s structure and that of other members of the ABC family is used to infer hints about the YenTc’s specificity and its dynamics. For example, the authors argue the structures of other members of the family may provide models of alternative conformational states that this complex may assume during thermal fluctuations or during the incorporation reaction. They infer a qualitative model for the dynamical mechanism through which the characteristic chitinase subunits of YenTc are incorporated into the macromolecular assembly. They also speculate that the YenTc prepore conformation found represents a pre-primed intermediate state in the pore forming process. Finally, the authors perform a filogenetic analysis to infer that this subclass of ABC toxins has been evolved relatively recently. Their phylogenetic analysis reveals specific evolutionary hotspots near the site which plays a key role in the incorporation process (namely, where the chitinase binding domains are inserted into the TcA unit).\nThis is certainly an extensive and detailed multi-disciplinary work, which skilfully combines experimental and bioinformatic techniques to provide new and relevant structural information, along with model for the key structural dynamics, cross-correlating this information with evolutionary insight. The paper is well written and documented. The figures are clear.\nCollectively, the body of work contained in this paper is certainly remarkable.\nAs a computational biophysics, I struggle to specifically assess the relevance of the CryEM results reported here for the structural biology community interested in ABC toxins, and to fully appreciate the technical challenge involved in resolving this specific structure by CryoEM.\nOn the other hand, I do see how establishing a mechanism for the incorporation of chitinase subunits in tho the macromolecular assembly would have very important implications. For example, it may inspire novel pharmacological strategies to develop YetTc inhibitors, e.g., by designing small molecules that bind with pockets that are only exposed by partially incorporated structures, arguably preventing the completion of the incorporation process.\nUnfortunately, while the structural results of this work are robust and well grounded into CryoEM experimental results, the inferred dynamical mechanism is admittedly qualitative and, most importantly, only speculative. In the lack of computational or experimental evidence to support the proposed mechanism, the paper in its present form would be mostly of interest for the structural biology community. A such, it would be more suitable for publication in a specialized journal.\nFor this work to become eligible for publication on an impactful generalist journal such as Nature Communications, the authors should substantiate the evidence in support of their dynamical insight. At the very least, they should detail a roadmap to outline an organized combination of feasible computational and experimental works that would ultimately lead to validating or disproving their model of mechanism. For example, given the large size of these macromolecular complexes and the complexity of the incorporation reaction, all-atom plain Molecular Dynamics simulations would obviously not be an option. However, probably simpler coarse grained models in which subunits are treated as semi-rigid domains via a ball-and-stick models may yield some important hints. Similarly, would time resolved optical techniques (such as FRET), HDX substitution, electron spray experiment be applicable to this system?\nIn conclusion, this work is remarkable and this manuscript would probably be publishable as is on a specialized journal. However, for publication on Nature Communication, I feel that more work is needed to expand and substantiate the claims about dynamics. I would be happy to review a revised version that takes significant steps in this direction.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "POINT-BY-POINT RESPONSE TO REVIEWER COMMENTS\nWe thank the reviewers for their positive feedback and helpful suggestions, which have aided us in preparing our improved manuscript for resubmission. Bellow we include a pointby-point response to the reviewers comments, with changes highlighted in the revised manuscript text.\nReviewer #1 (Remarks to the Author):\nReview of \"Complete structures of the YenTc holotoxin prepore and pore reveal the evolutionary basis for chitinase incorporation into ABC toxins\"\nThis manuscript presents a detailed structural analysis of the YenTc holotoxin from Yersinia entomophaga, using single-particle cryo-EM and AlphaFold2-assisted modeling to determine its architecture in both prepore and pore-forming states.\nThe authors provide a mechanistic explanation for chitinase incorporation within the toxin structure, supported by phylogenetic analyses that highlight its recent evolutionary emergence. The study significantly advances the understanding of ABC toxin diversity, structure, and function, providing key insights into their mode of action and evolutionary origins. However, additional validation and clarification are needed to justify publication in Nature Communications. Addressing the concerns regarding resolution, data collection, refinement protocols, and model validation will significantly strengthen the manuscript.\nMajor Strengths\n1. Structural Insights: The determination of both prepore and pore configurations provides a comprehensive view of YenTc's conformational transitions, enhancing our understanding of ABC toxin assembly and activation.\n2. Mechanistic Elucidation: The identification of chitinase-binding domains (CBD-A and CBDB) offers novel insights into protein-protein interactions within ABC toxins. The structural basis of chitinase incorporation into YenTc represents the first structural example of a novel protein/subunit recruitment mechanism, adding significant novelty beyond their previous work.\n3. Evolutionary Perspective: The phylogenetic analysis strengthens the manuscript by positioning YenTc within the broader ABC toxin family and revealing the evolutionary significance of chitinase incorporation.\nWe thank Reviewer one for their positive comments and note the concerns regarding additional validation and clarification which we have addressed in turn below.\nMajor revisions:\n1. Resolution limitations: Some structural elements, particularly the CBDA-Chi2 and CBDBChi1 interfaces, appear less resolved. The authors attempt to address this using hybrid modeling and combine it with MD and XL-MS to validate their structure experimentally. However, additional information should be provided to evaluate the structural quality and assess whether their claims are experimentally supported.\nWe acknowledge the concerns of the reviewer regarding model quality and address these in turn below. Importantly, we want to be clear in stating that only the CBD-B/Chi1 interface required the additional validation indicated by the reviewer above. CBD-A/Chi2 could be built purely using the cryo-EM data. We have reworded the manuscript to make this clear and address reasons why the map is less well resolved in the CBD-B/Chi1 region in response to various comments that follow.\nAccording to nature's reporting standards, official validation reports from the wwPDB are required for peer review of macromolecular structures (https://www.nature.com/natureportfolio/editorial-policies/reporting-standards). Please provide these reports for the structures deposited in the PDB (YenTc holotoxin prepore and pore structures).\nWe apologise that these reports were not available to the reviewer for initial review. We have now confirmed that these have been uploaded into the reviewing system and are available alongside the revised manuscript.\nAlso, a figure showing the cryo-EM map of CBDA-Chi2 and CBDB-Chi1 with their corresponding atomic models should be included in the main manuscript.\nIn our revised submission, we have included a new Figure S3 which shows the model-map fit for a number of key regions of the map, including the CBD-A/Chi2 interface. We note that model-map fit is also reported on in the PDB validation report, which was erroneously not included in the reviewer file in our original submission.\nIn-line with the reviewer’s comments about limitations of map quality, we have also revised Figure 4B so that the side chains of key interface residues are no longer shown. While the structure refinement protocol involves localised molecular dynamics (as is standard in ISOLDE) and therefore informs likely interactions that stabilise the interface, the map itself is of insufficient local resolution to precisely define side chain orientations in this region and we do not wish to give the unintended impression that this may be the case.\nWith respect to the CBD-B and Chi1 interface, we want to be clear that we did not use the cryo-EM data to model this specific interface. Building this part of the structure (and only this part of the structure) relied on AF2 structure predictions, validated by MD simulations and crosslinking MS. Evidence that structural dynamics (rather than dataset size of quality) impacts the resolvability of this region, and prevents modelling of CBD-B/Chi1 interface using cryo-EM data alone, is also provided by our new 3D variability analysis. This analysis is discussed in more detail in response to point 3 below.\n2. Structural quality concerns: Figure 2D shows that the secondary structure of the two blades undergoing major conformational changes is lost. In PTC3, each blade contains 3-4 beta-strands, suggesting that the refinement protocol used for YenTc may need revision, including correct secondary structure restraints if necessary.\nWe thank the reviewer for these comments. When we revisited the structure, we noticed that while the secondary structure does appear to have been lost, when overlayed with PTC3 the beta-strands are highly similar. This observation strongly justifies the application of secondary structure restraints as suggested, which we implemented during a subsequent model refinement. We found that this retained the secondary structure as expected and have updated Figure 2D, the PDB deposition and our PDB validation report to reflect the new model.\n3. Data collection concerns: The study collected only 616 and 1222 movies for the prepore and pore conformations, respectively. Could additional data collection (e.g., 10x more data) improve resolution and model quality? The authors should discuss this limitation and justify their data collection strategy.\nWe acknowledge the reviewer’s concerns with dataset size as it pertains to resolution and model quality, and their request for justification of our data collection strategy.\nWith respect to the data collection strategy, we included in our revised manuscript the following statement in the first paragraph of the Results: “Notably, all of the datasets used in the course of this work were collected manually using a side-entry cryo-holder on a Tecnai F30 FEG-TEM fitted with a K2 Summit direct electron detector.” This is significant, since collection of datasets the size of which we describe in this manuscript took several days of manual microscope operation per dataset, as is typical for this generation of microscope, and as opposed to the relatively short timeframes that would be required on the very latest and most state-of-the-art instruments.\nIn this regard, we note that there has been recent commentary around efforts to democratise access to Cryo-EM worldwide (e.g. doi:10.1038/nmeth.3946, doi:10.1126/sciadv.adr0438). Our study is therefore a noteworthy addition to this initiative, in that it stands as an example of how inexpensive and/or older generation cryo-EM instruments can still deliver impactful structural insights. We therefore included the following additional sentence in Results paragraph one: “Given that financial and geographical barriers often prohibit access to cutting edge, automated microscopes our results serve as an important example highlighting the capabilities of previous generation instruments and thus contribute to the “democratisation” of cryo-EM.”\nTo answer the question of whether a more extensive data collection strategy might improve the map resolution and quality we performed a 3D variability analysis of our dataset. This analysis is helpful in understanding the main sources of heterogeneity, as well as the extent to which a more extensive data collection is likely to improve the poorly resolved regions of the structure. The results of this analysis are summarised in Supplementary Movie 2, which shows that continuous structural heterogeneity limits the resolvability of the structure in some regions – primarily in the region of the CBD-B domain and the attached Chi1 subunit, and to a lesser in the region of the Chi2 subunit. Importantly, these are the two regions of the map where local resolution is lowset, and while 3D classification of very large (e.g. 10x larger datasets) has been shown to improve the resolution of flexible domains in some cases of discrete structural heterogeneity (i.e. a finite number of distinct conformational states), continuous heterogeneity is much more difficult to resolve. We therefore concluded that a more extensive data collection is unlikely to improve the map quality in the regions of greatest concern.\nFinally, we conducted a preliminary analysis of a much larger cryo-EM dataset that we have very recently collected on a modern instrument. This analysis yielded results entirely consistent with the conclusion we arrived at above i.e. the poorly resolved domains remain poorly resolved. We have chosen not to include this data in the current manuscript as it does not advance the main claims of the manuscript, which we feel are fully supported by the structural data already included. Moreover, inclusion of the data would detract from the aforementioned value of the work from the perspective of democratising access to cryo-EM. While the latter was not an original motivation for the study, it nonetheless stands as an exemplar for this important initiative.\nReviewer #2 (Remarks to the Author):\nIn review of the manuscript, \"Complete structures of the YenTc holotoxin prepore and pore reveal the evolutionary basis for chitinase incorporation into ABC toxins.\"\nThis study presents two low-resolution cryo-EM structures of YenTc, one in its holotoxin prepore complex form and the other in its putative ‘pore’ conformation. The authors used AlphaFold to model a previously unresolvable segment, the chitinase-binding domain (CBD), and used bioinformatics to further characterise the evolutionary history of these proteins. ABC toxins of Yersinia represent a fascinating class of functional proteins. However, we find that this manuscript offers only an incremental advance over previously reported structures of YenTc, raising concerns about its true novelty. While the resolution is an improvement over prior models, it remains relatively modest and still necessitates the use of Alphafold to complete the structural model (previous publications show the pore and prepore structures of TcA (Piper et al. (2019)), the complex of TcBC (Busby et al. (2013)), as well as 2D classes and models of the full YenTc holotoxin (Landsberg et al. (2014)).\nWe thank the reviewer for their comments, and acknowledge their concerns with regard to the limited resolution of the data presented in the manuscript, which we have already addressed in part in response to Reviewer 1 above.\nWe disagree strongly with the reviewer’s suggestion that improvements in the resolution of our models compared to previous studies are modest. This is most evident in the pore-form structure. The 2019 paper by Piper et al. cited by the reviewer above presents an 11 Å map of the TcA pre-pore (a shape with no resolvable secondary structure elements), which not only lacks the TcBC subunit, but is insufficient to allow any sort of structural interpretation beyond rigid body fitting. The structure reported here allows – for the first time - a detailed molecular interpretation of YenTc’s pre-pore to pore transition that until now has not been possible. We\nTo make this point clear, we have updated the manuscript in several places to emphasise improvements in resolution and (where appropriate) the enhanced mechanistic understanding these improvements facilitate.\nLine 84-86: Change “The latter most likely mimics the pore-forming configuration adopted in vivo, and therefore provides structural insights into the mechanism of pore formation.” To ”The latter in particular represents a substantial improvement over previously reported structures, and therefore provides new structural insights into the molecular transitions that accompany pore-formation.”\nLine 126-128: Added “... substantially improving on the structure we reported previously at a resolution of 11 Å and allowing detailed interpretation of the molecular transitions associated with pore-formation.”\nLine 190-196: Changed “The transition of YenTc from the pre-pore to the pore conformation is characterised by substantial conformational changes, that are almost exclusively confined to the TcA component and are therefore similar to those described (albeit in less detail) in our previous structure of the TcA component reconstituted in liposomes 13. The resolution of the TcA component in the nanodisc-embedded pore-form map is substantially improved, facilitating a more confident and complete positioning of the individual structural domains and allowing us to derive new insights into aspects of the pre-pore-to-pore transition that appear to distinguish YenTc from PTC3.”\nTo “The transition of YenTc from the prepore to the pore conformation is characterised by substantial conformational changes, that are almost exclusively confined to the TcA component. Whilst these changes are qualitatively similar to what was described in our previous manuscript that reported the structure of the TcA component reconstituted in liposomes 13, the resolution of this nanodisc-embedded holotoxin assembly is substantially improved, facilitating a more confident and complete positioning of the individual structural domains and allowing us to derive new insights into aspects of the prepore-to-pore transition.”\nThe reviewer notes that AlphaFold2 was required to complete the structural model, and while this is correct, we wish to ensure that the extent to which this was necessary is not over-stated. We therefore emphasise that only CBD-B/Chi1 was modelled with AF2 and this was necessitated by the high degree of continuous structural heterogeneity in this region, a limitation which is very likely to remain within any study of this system (see also response to Reviewer 1 comment 3, above). Indeed, it is not uncommon to resort to AF2-assisted modelling of flexible, otherwise unresolvable domains within cryo-EM structures and many recent examples exist within the literature (e.g. doi:10.1038/s41467-023-44670-5, doi:10.1038/s41467-023-37666-8, doi:10.1038/s41467-022-33175-2). Supplementary Movie 2 visualises and emphasises the inherent flexibility of the CBD-B/Chi1 region that necessitates hybrid model building approach. The graphical summary presented in Fig. S13 highlights the fact that the vast majority of the structure is modelled based purely on the cryo-EM data.\n####We appreciate that improvements have been made to the resolution from previous reports, but still believe the increase from 11 Å to 4 Å, 4.9 Å and 5.7 Å is still provides only a modest increase in the interpretive value, and therefore we still believe this work to be an incremental advance on previous work.\nDue to the limited data presented, the manuscript tends to over-discuss minor details, which impacts the overall readability and engagement of the text. This is a strong enough feature to make the results section read much like a discussion section in places.\nWe appreciate this feedback from the reviewer, and acknowledge that in places, the detailed structural analysis we present of this large complex goes into more detail than what might be considered necessary for a broad readership journal. In response, we have modified the text in several places, including but not limited to:\nLines 168-173\nLines 180-185\nLines 191-197\nLines 233-258 (in response to the specific point below)\nLines 308-310 (in response to the specific point below)\nAll changes have been highlighted in the revised manuscript and have been made with the goal in mind of enhancing readability and emphasising the key outcomes and conclusions from the current study.\n####We are satisfied with these edits\nThe informatics component, and statements regarding domain insertion and interaction (e.g. page 10 line 20 “may provide the first structural example of a novel protein/subunit recruitment mechanism”) are essentially true of ANY system and are descriptive of natural variation rather than any especially interesting mechanism.\nWe agree with the reviewer’s interpretation and have revised this statement to instead emphasise our conclusion that the Chi2-CBD-A interface is adapted from polymeric pilin proteins.\nLine 308-310: Changed “We were unable to find any structural homologues of this interface, suggesting that the interaction identified between Chi2NTD and CBD-A may provide the first structural example of a novel protein/subunit recruitment mechanism.”\nTo “Thus, the mechanism of polymeric pilin assembly appears to have been co-opted here to facilitate recruitment of Chi2 into a stable complex with YenTc.”\n####We are satisfied with these edits\nA similar scenario exists in the over-documentation of the linker helix extension – there is not enough importance in essentially just discussing minor variations.\nAs noted above, we agree broadly with the reviewer’s concerns that the manuscript could be made more concise in some areas, and have significantly modified the discussion of the linker helix with this goal in mind. We do believe that this aspect of the structure is crucial to explaining differences in the previously published PTC3 and YenTc (our study) pore form structures. We therefore retained a more concise structural analysis (lines 204-258) in the main text and moved some of the more detailed aspects of our analysis – which nonetheless substantiate our conclusions – to the Supplementary text.\n####We are satisfied with these edits\nMinor points:\nAbstract – “we determine structures …using cryo-EM and Alphafold2-assisted structural modelling”; determination (experimental) and modelling (in silico) should not be conflated/linked in this manner and require separating or rewording\nLine 30-32: Changed “Here we determine structures of the YenTc holotoxin assembly in both prepore and pore-forming configurations using cryo-EM and Alphafold2-assisted structural modelling.\nTo “Here we determine structures of the YenTc holotoxin assembly in both prepore and pore-forming configurations using cryo-EM in conjunction with Alphafold2-assisted structural modelling of flexible domains.”\n####We are satisfied with these edits\nLine 67: “pentameric structure” – Describing TcA as pentameric is incorrect as its is not 5 polypeptides, but a c5 symmetric icosmeric complex made of a 4-sub unit protamer.\nChanged to “Each polypeptide chain is present in five copies, yielding a 2.1 MDa complex.”\n####We are satisfied with these edits\nLine 74: “juxtaposed” – The usage of this word is incorrect as its used to highlight contrast between two adjacent objects.\nThe sentence now reads: “Membrane engagement in vivo is likely to involve interaction with host-specific cell surface receptors that position the complex so that the tip is driven through an adjacent membrane bilayer.”\n####We are satisfied with these edits\nLine 91: Chitinase binding domain (CBD) is a poor choice of acronym, as it is highly used, including ‘carbohydrate binding domain’, and the very similar ‘chitin binding domain’.\nWe acknowledge the reviewer’s concern here, and in preparing our initial manuscript we did consider several alternative acronyms. Initially, we used “RBD-like” domains, but we preferred the term “chitinase-binding domain” since this more accurately reflects their demonstrated functional role. We note that while carbohydrate binding domains and chitin binding domains do already exist (and thus, there is a literature precedent for using the same acronym for different domains), neither of these is referred to in our manuscript and thus the acronym is not ambiguous in the context of our manuscript. Having said this, we have no strong preference for an acronym here and are happy to replace it with an alternative if this is deemed essential.\n####This is not essential\nFigure 1 AC: should the cream coloured chitinase be yellow as its part of the yellow protamer?\nWe used a lighter colour for the chitinase densities associated with each protomer to distinguish them from the main shell, reflecting the fact that they are separate polypeptide chains. The choice of cream was made for the chitinase in question as a lighter shade of yellow made the chitinase difficult to visualise on a white background.\n####We are satisfied with this\nFigure 2A-E: there is a substantial conformational change in the protein, however there is no evidence of the density that was used to support this. Is this region an Alphafold model or was it morphed from the closed structure? As the local resolution appears to be between 5-7 Å, it would be good to have figures of the model and associated density in the supplementary.\nWe thank the reviewer for their comment. In response, we note that this region was rebuilt into the EM map, guided by structural similarities observed when comparison was made to the PTC3 holotoxin. In response to comments also made by Reviewer 1, we included a new supplementary figure (Figure S3) that illustrates the model map fit in various regions of the structure, and have incorporated this region into the figure as panel C.\nAs noted in an earlier response, the only region of the structure modelled by AlphaFold2 was the Chi1:CBD-B interface (Figure S13).\n####We are satisfied with these edits\nLine 271-273 and line 323-325: the first states the “poor local map resolution had until now prevented us from building structures”, suggesting that the resolution now allows building of the model. However the second section states “The resolution of our maps in the region of the Chi1 region is more modest…while this prevented de novo building, a hybrid model building approach [with AlphaFold2] proved successful”. These statements are contradictory and the first perhaps suggests that the data are better than they are and so requires toning down\nWe thank the reviewer for their comment and can appreciate the contradictory nature of these statements, which was unintentional on our part. To avoid confusion, we have amended the main text as follows:\nLine 261-263: Changed “poor local map resolution had until now prevented us from building structures of the Chi1/YenA2 and Chi2/YenA1 interfaces.”\nTo “poor local map resolution meant we had been unable to resolve the Chi2-YenA1 and Chi1-YenA2 interfaces.”\nWe also updated the text that follows to clearly define which regions can now be modelled de novo, and which cannot.\nLine 265-270: Changed “In this study, we could resolve two protrusions from the surrounding α-helical shell per protomer which clearly establish interfaces with the chitinase subunits. We named these newly resolved domains chitinase-binding domains (CBDs), reflecting their structural roles (Fig. 4A). CBD-A (YenA1 residues 311-472) forms the major binding interface with the Chi2 subunit of TcA, while CBD-B (YenA2 residues 400-520) forms the major binding interface with the Chi1 subunit.”\nTo: “The improved maps reported here allow unequivocal identification of two previously unresolved chitinase-binding domains (CBD) inserted into the TcA shell (Fig. 4A). CBD-A (YenA1 residues 311-472), forms the major binding interface with the Chi2 subunit of TcA and could be modelled based on the map density alone. CBD-B (YenA2 residues 400-520) forms the major binding interface with the Chi1 subunit but due to substantial conformational dynamics, required a hybrid modelling approach (detailed below).”\nNoting the reviewer’s concerns about resolvability and model quality, we also added the following explanation of why the Chi1 interface required a hybrid model building approach.\nLine 312-322: Changed “The resolution of our maps in the region of the Chi1 interface is more modest, likely due to substantial conformational dynamics noted in this work and prior studies. While this prevented de novo model building, a hybrid model building approach proved successful, combining structure prediction using AlphaFold2 multimer (AF2m), with cross-linking mass spectrometry (XL-MS), MD simulations and structural data derived from our previously determined crystal structure of the Chi1 subunit.”\nTo ”A 3D variability analysis using our cryo-EM data confirmed that substantial conformational dynamics (Movie S2) are the most likely contributing factor to the poor local map resolution in the region of the Chi1:CBD-B interface, prohibiting high resolution structure determination. To model this interface, we therefore employed a hybrid model building approach using AlphaFold2 multimer (AF2m), cross-linking mass spectrometry (XLMS), MD simulations and structural data derived from our previously determined crystal structure of the Chi1 subunit.”\n####We are satisfied with these edits\nFigure 2 legend should read “TcB and TcC are in pink”\nWe have corrected the figure legend.\n####We are satisfied with these edits\nFigure 4 line 285: RBD-like domains – should be referred to as CBD domains for continuity.\nWe have corrected the figure legend.\n####We are satisfied with these edits\nReviewer #3 (Remarks to the Author):\nThis work focuses on the structural biology of YenTc, a representative element of a subclass of the ABC toxins. The mechanism of action of these toxins has not yet been fully clarified. While previous work from the authors yielded the structure of the “binding” and the “active” subunits of this macromolecular complex, the present work provides the missing structural details, including models for the prepore and the pore forming structures.These are resolved by combining cryoEM experiments with bioinformatic-based structural modeling (via Alphafold-2).\nWe thank the reviewer for highlighting the significance of our work in filling gaps in our otherwise incomplete knowledge of these systems.\nThe difference between the YenTc’s structure and that of other members of the ABC family is used to infer hints about the YenTc’s specificity and its dynamics. For example, the authors argue the structures of other members of the family may provide models of alternative conformational states that this complex may assume during thermal fluctuations or during the incorporation reaction. They infer a qualitative model for the dynamical mechanism through which the characteristic chitinase subunits of YenTc are incorporated into the macromolecular assembly.\nWe thank the reviewer for recognising our efforts to highlight structural differences between YenTc and other ABC toxin family members, and the potential this offers to provide insights into aspects of their mechanism.\nFor the absolute avoidance of confusion, we do wish to clarify that we are not proposing a ‘dynamical mechanism’ of chitinase incorporation. This is elaborated on in response to a subsequent comment from Reviewer 3 below.\nThey also speculate that the YenTc prepore conformation found represents a pre-primed intermediate state in the pore forming process. Finally, the authors perform a filogenetic analysis to infer that this subclass of ABC toxins has been evolved relatively recently. Their phylogenetic analysis reveals specific evolutionary hotspots near the site which plays a key role in the incorporation process (namely, where the chitinase binding domains are inserted into the TcA unit).\nAgain, we thank the reviewer for their positive summary of the phylogenetic analysis in this study.\nThis is certainly an extensive and detailed multi-disciplinary work, which skilfully combines experimental and bioinformatic techniques to provide new and relevant structural information, along with model for the key structural dynamics, cross-correlating this information with evolutionary insight. The paper is well written and documented. The figures are clear.\nCollectively, the body of work contained in this paper is certainly remarkable.\nWe thank the reviewer for their enthusiastic review of our manuscript and appreciate their positive remarks on the body of work as a whole.\nAs a computational biophysics, I struggle to specifically assess the relevance of the CryEM results reported here for the structural biology community interested in ABC toxins, and to fully appreciate the technical challenge involved in resolving this specific structure by CryoEM.\nIn combination with the concerns of Reviewer 2, we do recognise that parts of our manuscript could be refined to improve accessibility and clarity. We believe that the changes made in response to Reviewer 2 have also helped with highlighting the significance of our study to a broader audience.\nOn the other hand, I do see how establishing a mechanism for the incorporation of chitinase subunits in tho the macromolecular assembly would have very important implications. For example, it may inspire novel pharmacological strategies to develop YetTc inhibitors, e.g., by designing small molecules that bind with pockets that are only exposed by partially incorporated structures, arguably preventing the completion of the incorporation process.\nUnfortunately, while the structural results of this work are robust and well grounded into CryoEM experimental results, the inferred dynamical mechanism is admittedly qualitative and, most importantly, only speculative. In the lack of computational or experimental evidence to support the proposed mechanism, the paper in its present form would be mostly of interest for the structural biology community. A such, it would be more suitable for publication in a specialized journal.\nWe acknowledge these comments alongside those made previously by his reviewer about a “dynamical mechanism” of chitinase incorporation, and wish to address the apparent confusion that may have arisen in this regard.\nWe stress that our studies do not provide evidence for, nor were we implying that they support, a mechanism whereby chitinases are dynamically incorporated into the structure of YenTc.\nYenTc is however unique amongst ABC toxins, in that it is the only structural characterised example that includes chitinase subunits. Our combined phylogenetic and structural analysis provides evidence for a plausible evolutionary mechanism via which RBD or RBD-like domains in the TcA shell were modified or substituted, conferring the ability to include chitinases. These chitinases have ultimately migrated into the same operon as other components of YenTc, and are an essential structural component of the YenTc complex. Indeed, all evidence suggests the chitinase subunits remain tightly associated with YenTc, from the point of secretion through to receptor binding and pore formation. The dynamic aspect of the mechanism is in relation to structural motion, rather than an “on/off” mechanisms of incorporation, which the reviewer may have mistakenly thought we were implying?\nIn response to comments made by other reviewers, we performed a 3D variability analysis of our existing data sets, which is entirely consistent with the proposed dynamics that we attribute to the chitinase binding domains and attached chitinase subunits (Movie S2). Thus, we believe our claims of structural dynamics are already well supported.\nHaving critically self-evaluated the manuscript, we can appreciate that some parts of our original submission could be improved in order to try and avoid a similar misunderstanding by other readers. To address this, we have additionally modified Figure S7 to more clearly emphasise that this is a structural comparison between the CfaE-AD system and CBD-A, and thus provides a plausible mechanism that occurs on an evolutionary timescale, rather than structural dynamics or rearrangements within the relevant domain of YenTc.\nWe have also substantially modified Figure S10, in an effort to achieve enhanced clarity of the structural dynamics implied by the pre-pore to pore transitions of both YenTc and PTC3.\nThis revised figure captures structural data from several previous studies, as well as the novel structural data reported in our current manuscript. Importantly, it highlights that the pre-pore structure of YenTc is more similar to a recently described PTC3 intermediate state. There is no evidence that YenTc adopts a configuration similar to the PTC3 prepore, which would require a decoupling of the chitinase subunits, a message that we accept may have been poorly conveyed in our previous version of Figure S10 and that may have contributed to the underlying confusion.\nFor this work to become eligible for publication on an impactful generalist journal such as Nature Communications, the authors should substantiate the evidence in support of their dynamical insight. At the very least, they should detail a roadmap to outline an organized combination of feasible computational and experimental works that would ultimately lead to validating or disproving their model of mechanism. For example, given the large size of these macromolecular complexes and the complexity of the incorporation reaction, all-atom plain Molecular Dynamics simulations would obviously not be an option. However, probably simpler coarse grained models in which subunits are treated as semi-rigid domains via a ball-and-stick models may yield some important hints. Similarly, would time resolved optical techniques (such as FRET), HDX substitution, electron spray experiment be applicable to this system?\nWe thank the reviewer for these suggestions, but feel that having clarified the confusion around our implied mechanism, these experiments are unnecessary to substantiate the main claims of the manuscript. Coincidentally we have already conducted all atom MD simulations of this system as part of a separate study which looks at an entirely different aspect of the YenTc’s function. The key findings of this study remain to be fully validated, but are nonetheless unrelated to the current body of work reported in this manuscript and do not in any way advance (or contradict) the findings described in the current manuscript. Indeed, we believe that all of the conclusions reached in this manuscript are adequately and completely supported by the experimental evidence already supplied in our revised manuscript.\nIn conclusion, this work is remarkable and this manuscript would probably be publishable as is on a specialized journal. However, for publication on Nature Communication, I feel that more work is needed to expand and substantiate the claims about dynamics. I would be happy to review a revised version that takes significant steps in this direction.\nWe appreciate the concluding remarks. We hope that having provided clarification with regards to our claims and updating the manuscript in several places to improve clarity and accessibility for broader audiences, the Reviewer will agree that our claims are well substantiated by the evidence provided in the revised manuscript. We would be happy to work with the Reviewer to address any residual concerns.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I have reviewed the revised manuscript and the authors' responses to my initial comments. I am pleased to report that the authors have comprehensively addressed my major concerns regarding resolution limitations, structural quality, and data collection strategy. They provided the requested wwPDB reports and a new Figure S3 to validate map-model fit, clarifying that AlphaFold2 modeling was applied only to intrinsically flexible regions like the CBD-B/Chi1 interface, supported by MD and XL-MS data. The issue of lost secondary structure in TcB blades (Figure 2D) was resolved through re-refinement with appropriate restraints. Furthermore, the authors provided a robust justification for their data collection strategy, explaining the practicalities of manual collection on older equipment and demonstrating through 3D variability analysis that intrinsic structural heterogeneity, rather than data quantity, was the primary limitation to higher resolution in certain areas. These revisions significantly enhance the manuscript's rigor and transparency.", "reviewer_2": "Our reviewer points prefaced by #### below\nReviewer #2 (Remarks to the Author):\nIn review of the manuscript, \"Complete structures of the YenTc holotoxin prepore and pore reveal the evolutionary basis for chitinase incorporation into ABC toxins.\"\nThis study presents two low-resolution cryo-EM structures of YenTc, one in its holotoxin prepore complex form and the other in its putative ‘pore’ conformation. The authors used AlphaFold to model a previously unresolvable segment, the chitinase-binding domain (CBD), and used bioinformatics to further characterise the evolutionary history of these proteins. ABC toxins of Yersinia represent a fascinating class of functional proteins. However, we find that this manuscript offers only an incremental advance over previously reported structures of YenTc, raising concerns about its true novelty. While the resolution is an improvement over prior models, it remains relatively modest and still necessitates the use of Alphafold to complete the structural model (previous publications show the pore and prepore structures of TcA (Piper et al. (2019)), the complex of TcBC (Busby et al. (2013)), as well as 2D classes and models of the full YenTc holotoxin (Landsberg et al. (2014)).\nWe thank the reviewer for their comments, and acknowledge their concerns with regard to the limited resolution of the data presented in the manuscript, which we have already addressed in part in response to Reviewer 1 above.\nWe disagree strongly with the reviewer’s suggestion that improvements in the resolution of our models compared to previous studies are modest. This is most evident in the pore-form structure. The 2019 paper by Piper et al. cited by the reviewer above presents an 11 Å map of the TcA pre-pore (a shape with no resolvable secondary structure elements), which not only lacks the TcBC subunit, but is insufficient to allow any sort of structural interpretation beyond rigid body fitting. The structure reported here allows – for the first time - a detailed molecular interpretation of YenTc’s pre-pore to pore transition that until now has not been possible. We\nTo make this point clear, we have updated the manuscript in several places to emphasise improvements in resolution and (where appropriate) the enhanced mechanistic understanding these improvements facilitate.\nLine 84-86: Change “The latter most likely mimics the pore-forming configuration adopted in vivo, and therefore provides structural insights into the mechanism of pore formation.” To ”The latter in particular represents a substantial improvement over previously reported structures, and therefore provides new structural insights into the molecular transitions that accompany pore-formation.”\nLine 126-128: Added “... substantially improving on the structure we reported previously at a resolution of 11 Å and allowing detailed interpretation of the molecular transitions associated with pore-formation.”\nLine 190-196: Changed “The transition of YenTc from the pre-pore to the pore conformation is characterised by substantial conformational changes, that are almost exclusively confined to the TcA component and are therefore similar to those described (albeit in less detail) in our previous structure of the TcA component reconstituted in liposomes 13. The resolution of the TcA component in the nanodisc-embedded pore-form map is substantially improved, facilitating a more confident and complete positioning of the individual structural domains and allowing us to derive new insights into aspects of the pre-pore-to-pore transition that appear to distinguish YenTc from PTC3.”\nTo “The transition of YenTc from the prepore to the pore conformation is characterised by substantial conformational changes, that are almost exclusively confined to the TcA component. Whilst these changes are qualitatively similar to what was described in our previous manuscript that reported the structure of the TcA component reconstituted in liposomes 13, the resolution of this nanodisc-embedded holotoxin assembly is substantially improved, facilitating a more confident and complete positioning of the individual structural domains and allowing us to derive new insights into aspects of the prepore-to-pore transition.”\nThe reviewer notes that AlphaFold2 was required to complete the structural model, and while this is correct, we wish to ensure that the extent to which this was necessary is not over-stated. We therefore emphasise that only CBD-B/Chi1 was modelled with AF2 and this was necessitated by the high degree of continuous structural heterogeneity in this region, a limitation which is very likely to remain within any study of this system (see also response to Reviewer 1 comment 3, above). Indeed, it is not uncommon to resort to AF2-assisted modelling of flexible, otherwise unresolvable domains within cryo-EM structures and many recent examples exist within the literature (e.g. doi:10.1038/s41467-023-44670-5, doi:10.1038/s41467-023-37666-8, doi:10.1038/s41467-022-33175-2). Supplementary Movie 2 visualises and emphasises the inherent flexibility of the CBD-B/Chi1 region that necessitates hybrid model building approach. The graphical summary presented in Fig. S13 highlights the fact that the vast majority of the structure is modelled based purely on the cryo-EM data.\n####We appreciate that improvements have been made to the resolution from previous reports, but still believe the increase from 11 Å to 4 Å, 4.9 Å and 5.7 Å is still provides only a modest increase in the interpretive value, and therefore we still believe this work to be an incremental advance on previous work.\nDue to the limited data presented, the manuscript tends to over-discuss minor details, which impacts the overall readability and engagement of the text. This is a strong enough feature to make the results section read much like a discussion section in places.\nWe appreciate this feedback from the reviewer, and acknowledge that in places, the detailed structural analysis we present of this large complex goes into more detail than what might be considered necessary for a broad readership journal. In response, we have modified the text in several places, including but not limited to:\nLines 168-173\nLines 180-185\nLines 191-197\nLines 233-258 (in response to the specific point below)\nLines 308-310 (in response to the specific point below)\nAll changes have been highlighted in the revised manuscript and have been made with the goal in mind of enhancing readability and emphasising the key outcomes and conclusions from the current study.\n####We are satisfied with these edits\nThe informatics component, and statements regarding domain insertion and interaction (e.g. page 10 line 20 “may provide the first structural example of a novel protein/subunit recruitment mechanism”) are essentially true of ANY system and are descriptive of natural variation rather than any especially interesting mechanism.\nWe agree with the reviewer’s interpretation and have revised this statement to instead emphasise our conclusion that the Chi2-CBD-A interface is adapted from polymeric pilin proteins.\nLine 308-310: Changed “We were unable to find any structural homologues of this interface, suggesting that the interaction identified between Chi2NTD and CBD-A may provide the first structural example of a novel protein/subunit recruitment mechanism.”\nTo “Thus, the mechanism of polymeric pilin assembly appears to have been co-opted here to facilitate recruitment of Chi2 into a stable complex with YenTc.”\n####We are satisfied with these edits\nA similar scenario exists in the over-documentation of the linker helix extension – there is not enough importance in essentially just discussing minor variations.\nAs noted above, we agree broadly with the reviewer’s concerns that the manuscript could be made more concise in some areas, and have significantly modified the discussion of the linker helix with this goal in mind. We do believe that this aspect of the structure is crucial to explaining differences in the previously published PTC3 and YenTc (our study) pore form structures. We therefore retained a more concise structural analysis (lines 204-258) in the main text and moved some of the more detailed aspects of our analysis – which nonetheless substantiate our conclusions – to the Supplementary text.\n####We are satisfied with these edits\nMinor points:\nAbstract – “we determine structures …using cryo-EM and Alphafold2-assisted structural modelling”; determination (experimental) and modelling (in silico) should not be conflated/linked in this manner and require separating or rewording\nLine 30-32: Changed “Here we determine structures of the YenTc holotoxin assembly in both prepore and pore-forming configurations using cryo-EM and Alphafold2-assisted structural modelling.\nTo “Here we determine structures of the YenTc holotoxin assembly in both prepore and pore-forming configurations using cryo-EM in conjunction with Alphafold2-assisted structural modelling of flexible domains.”\n####We are satisfied with these edits\nLine 67: “pentameric structure” – Describing TcA as pentameric is incorrect as its is not 5 polypeptides, but a c5 symmetric icosmeric complex made of a 4-sub unit protamer.\nChanged to “Each polypeptide chain is present in five copies, yielding a 2.1 MDa complex.”\n####We are satisfied with these edits\nLine 74: “juxtaposed” – The usage of this word is incorrect as its used to highlight contrast between two adjacent objects.\nThe sentence now reads: “Membrane engagement in vivo is likely to involve interaction with host-specific cell surface receptors that position the complex so that the tip is driven through an adjacent membrane bilayer.”\n####We are satisfied with these edits\nLine 91: Chitinase binding domain (CBD) is a poor choice of acronym, as it is highly used, including ‘carbohydrate binding domain’, and the very similar ‘chitin binding domain’.\nWe acknowledge the reviewer’s concern here, and in preparing our initial manuscript we did consider several alternative acronyms. Initially, we used “RBD-like” domains, but we preferred the term “chitinase-binding domain” since this more accurately reflects their demonstrated functional role. We note that while carbohydrate binding domains and chitin binding domains do already exist (and thus, there is a literature precedent for using the same acronym for different domains), neither of these is referred to in our manuscript and thus the acronym is not ambiguous in the context of our manuscript. Having said this, we have no strong preference for an acronym here and are happy to replace it with an alternative if this is deemed essential.\n####This is not essential\nFigure 1 AC: should the cream coloured chitinase be yellow as its part of the yellow protamer?\nWe used a lighter colour for the chitinase densities associated with each protomer to distinguish them from the main shell, reflecting the fact that they are separate polypeptide chains. The choice of cream was made for the chitinase in question as a lighter shade of yellow made the chitinase difficult to visualise on a white background.\n####We are satisfied with this\nFigure 2A-E: there is a substantial conformational change in the protein, however there is no evidence of the density that was used to support this. Is this region an Alphafold model or was it morphed from the closed structure? As the local resolution appears to be between 5-7 Å, it would be good to have figures of the model and associated density in the supplementary.\nWe thank the reviewer for their comment. In response, we note that this region was rebuilt into the EM map, guided by structural similarities observed when comparison was made to the PTC3 holotoxin. In response to comments also made by Reviewer 1, we included a new supplementary figure (Figure S3) that illustrates the model map fit in various regions of the structure, and have incorporated this region into the figure as panel C.\nAs noted in an earlier response, the only region of the structure modelled by AlphaFold2 was the Chi1:CBD-B interface (Figure S13).\n####We are satisfied with these edits\nLine 271-273 and line 323-325: the first states the “poor local map resolution had until now prevented us from building structures”, suggesting that the resolution now allows building of the model. However the second section states “The resolution of our maps in the region of the Chi1 region is more modest…while this prevented de novo building, a hybrid model building approach [with AlphaFold2] proved successful”. These statements are contradictory and the first perhaps suggests that the data are better than they are and so requires toning down\nWe thank the reviewer for their comment and can appreciate the contradictory nature of these statements, which was unintentional on our part. To avoid confusion, we have amended the main text as follows:\nLine 261-263: Changed “poor local map resolution had until now prevented us from building structures of the Chi1/YenA2 and Chi2/YenA1 interfaces.”\nTo “poor local map resolution meant we had been unable to resolve the Chi2-YenA1 and Chi1-YenA2 interfaces.”\nWe also updated the text that follows to clearly define which regions can now be modelled de novo, and which cannot.\nLine 265-270: Changed “In this study, we could resolve two protrusions from the surrounding α-helical shell per protomer which clearly establish interfaces with the chitinase subunits. We named these newly resolved domains chitinase-binding domains (CBDs), reflecting their structural roles (Fig. 4A). CBD-A (YenA1 residues 311-472) forms the major binding interface with the Chi2 subunit of TcA, while CBD-B (YenA2 residues 400-520) forms the major binding interface with the Chi1 subunit.”\nTo: “The improved maps reported here allow unequivocal identification of two previously unresolved chitinase-binding domains (CBD) inserted into the TcA shell (Fig. 4A). CBD-A (YenA1 residues 311-472), forms the major binding interface with the Chi2 subunit of TcA and could be modelled based on the map density alone. CBD-B (YenA2 residues 400-520) forms the major binding interface with the Chi1 subunit but due to substantial conformational dynamics, required a hybrid modelling approach (detailed below).”\nNoting the reviewer’s concerns about resolvability and model quality, we also added the following explanation of why the Chi1 interface required a hybrid model building approach.\nLine 312-322: Changed “The resolution of our maps in the region of the Chi1 interface is more modest, likely due to substantial conformational dynamics noted in this work and prior studies. While this prevented de novo model building, a hybrid model building approach proved successful, combining structure prediction using AlphaFold2 multimer (AF2m), with cross-linking mass spectrometry (XL-MS), MD simulations and structural data derived from our previously determined crystal structure of the Chi1 subunit.”\nTo ”A 3D variability analysis using our cryo-EM data confirmed that substantial conformational dynamics (Movie S2) are the most likely contributing factor to the poor local map resolution in the region of the Chi1:CBD-B interface, prohibiting high resolution structure determination. To model this interface, we therefore employed a hybrid model building approach using AlphaFold2 multimer (AF2m), cross-linking mass spectrometry (XLMS), MD simulations and structural data derived from our previously determined crystal structure of the Chi1 subunit.”\n####We are satisfied with these edits\nFigure 2 legend should read “TcB and TcC are in pink”\nWe have corrected the figure legend.\n####We are satisfied with these edits\nFigure 4 line 285: RBD-like domains – should be referred to as CBD domains for continuity.\nWe have corrected the figure legend.\n####We are satisfied with these edits\nWe thank the reviewer for their additional suggestions and we are pleased that we were able to satisfy all concerns flagged by the reviewer, outside of the perceived interpretative value of the structural data. We trust that our collective responses to this single point of concern through both rounds of revision have adequately defended the value of our work.", "reviewer_3": "This work focuses on the structural biology of YenTc, a representative element of a subclass of the ABC toxins. The mechanism of action of these toxins has not yet been fully clarified. While previous work from the authors yielded the structure of the “binding” and the “active” subunits of this macromolecular complex, the present work provides the missing structural details, including models for the prepore and the pore forming structures.These are resolved by combining cryoEM experiments with bioinformatic-based structural modeling (via Alphafold-2).\nWe thank the reviewer for highlighting the significance of our work in filling gaps in our otherwise incomplete knowledge of these systems.\nThe difference between the YenTc’s structure and that of other members of the ABC family is used to infer hints about the YenTc’s specificity and its dynamics. For example, the authors argue the structures of other members of the family may provide models of alternative conformational states that this complex may assume during thermal fluctuations or during the incorporation reaction. They infer a qualitative model for the dynamical mechanism through which the characteristic chitinase subunits of YenTc are incorporated into the macromolecular assembly.\nWe thank the reviewer for recognising our efforts to highlight structural differences between YenTc and other ABC toxin family members, and the potential this offers to provide insights into aspects of their mechanism.\nFor the absolute avoidance of confusion, we do wish to clarify that we are not proposing a ‘dynamical mechanism’ of chitinase incorporation. This is elaborated on in response to a subsequent comment from Reviewer 3 below.\nThey also speculate that the YenTc prepore conformation found represents a pre-primed intermediate state in the pore forming process. Finally, the authors perform a filogenetic analysis to infer that this subclass of ABC toxins has been evolved relatively recently. Their phylogenetic analysis reveals specific evolutionary hotspots near the site which plays a key role in the incorporation process (namely, where the chitinase binding domains are inserted into the TcA unit).\nAgain, we thank the reviewer for their positive summary of the phylogenetic analysis in this study.\nThis is certainly an extensive and detailed multi-disciplinary work, which skilfully combines experimental and bioinformatic techniques to provide new and relevant structural information, along with model for the key structural dynamics, cross-correlating this information with evolutionary insight. The paper is well written and documented. The figures are clear.\nCollectively, the body of work contained in this paper is certainly remarkable.\nWe thank the reviewer for their enthusiastic review of our manuscript and appreciate their positive remarks on the body of work as a whole.\nAs a computational biophysics, I struggle to specifically assess the relevance of the CryEM results reported here for the structural biology community interested in ABC toxins, and to fully appreciate the technical challenge involved in resolving this specific structure by CryoEM.\nIn combination with the concerns of Reviewer 2, we do recognise that parts of our manuscript could be refined to improve accessibility and clarity. We believe that the changes made in response to Reviewer 2 have also helped with highlighting the significance of our study to a broader audience.\nOn the other hand, I do see how establishing a mechanism for the incorporation of chitinase subunits in tho the macromolecular assembly would have very important implications. For example, it may inspire novel pharmacological strategies to develop YetTc inhibitors, e.g., by designing small molecules that bind with pockets that are only exposed by partially incorporated structures, arguably preventing the completion of the incorporation process.\nUnfortunately, while the structural results of this work are robust and well grounded into CryoEM experimental results, the inferred dynamical mechanism is admittedly qualitative and, most importantly, only speculative. In the lack of computational or experimental evidence to support the proposed mechanism, the paper in its present form would be mostly of interest for the structural biology community. A such, it would be more suitable for publication in a specialized journal.\nWe acknowledge these comments alongside those made previously by his reviewer about a “dynamical mechanism” of chitinase incorporation, and wish to address the apparent confusion that may have arisen in this regard.\nWe stress that our studies do not provide evidence for, nor were we implying that they support, a mechanism whereby chitinases are dynamically incorporated into the structure of YenTc.\nYenTc is however unique amongst ABC toxins, in that it is the only structural characterised example that includes chitinase subunits. Our combined phylogenetic and structural analysis provides evidence for a plausible evolutionary mechanism via which RBD or RBD-like domains in the TcA shell were modified or substituted, conferring the ability to include chitinases. These chitinases have ultimately migrated into the same operon as other components of YenTc, and are an essential structural component of the YenTc complex. Indeed, all evidence suggests the chitinase subunits remain tightly associated with YenTc, from the point of secretion through to receptor binding and pore formation. The dynamic aspect of the mechanism is in relation to structural motion, rather than an “on/off” mechanisms of incorporation, which the reviewer may have mistakenly thought we were implying?\nIn response to comments made by other reviewers, we performed a 3D variability analysis of our existing data sets, which is entirely consistent with the proposed dynamics that we attribute to the chitinase binding domains and attached chitinase subunits (Movie S2). Thus, we believe our claims of structural dynamics are already well supported.\nHaving critically self-evaluated the manuscript, we can appreciate that some parts of our original submission could be improved in order to try and avoid a similar misunderstanding by other readers. To address this, we have additionally modified Figure S7 to more clearly emphasise that this is a structural comparison between the CfaE-AD system and CBD-A, and thus provides a plausible mechanism that occurs on an evolutionary timescale, rather than structural dynamics or rearrangements within the relevant domain of YenTc.\nWe have also substantially modified Figure S10, in an effort to achieve enhanced clarity of the structural dynamics implied by the pre-pore to pore transitions of both YenTc and PTC3.\nThis revised figure captures structural data from several previous studies, as well as the novel structural data reported in our current manuscript. Importantly, it highlights that the pre-pore structure of YenTc is more similar to a recently described PTC3 intermediate state. There is no evidence that YenTc adopts a configuration similar to the PTC3 prepore, which would require a decoupling of the chitinase subunits, a message that we accept may have been poorly conveyed in our previous version of Figure S10 and that may have contributed to the underlying confusion.\nFor this work to become eligible for publication on an impactful generalist journal such as Nature Communications, the authors should substantiate the evidence in support of their dynamical insight. At the very least, they should detail a roadmap to outline an organized combination of feasible computational and experimental works that would ultimately lead to validating or disproving their model of mechanism. For example, given the large size of these macromolecular complexes and the complexity of the incorporation reaction, all-atom plain Molecular Dynamics simulations would obviously not be an option. However, probably simpler coarse grained models in which subunits are treated as semi-rigid domains via a ball-and-stick models may yield some important hints. Similarly, would time resolved optical techniques (such as FRET), HDX substitution, electron spray experiment be applicable to this system?\nWe thank the reviewer for these suggestions, but feel that having clarified the confusion around our implied mechanism, these experiments are unnecessary to substantiate the main claims of the manuscript. Coincidentally we have already conducted all atom MD simulations of this system as part of a separate study which looks at an entirely different aspect of the YenTc’s function. The key findings of this study remain to be fully validated, but are nonetheless unrelated to the current body of work reported in this manuscript and do not in any way advance (or contradict) the findings described in the current manuscript. Indeed, we believe that all of the conclusions reached in this manuscript are adequately and completely supported by the experimental evidence already supplied in our revised manuscript.\nIn conclusion, this work is remarkable and this manuscript would probably be publishable as is on a specialized journal. However, for publication on Nature Communication, I feel that more work is needed to expand and substantiate the claims about dynamics. I would be happy to review a revised version that takes significant steps in this direction.\nWe appreciate the concluding remarks. We hope that having provided clarification with regards to our claims and updating the manuscript in several places to improve clarity and accessibility for broader audiences, the Reviewer will agree that our claims are well substantiated by the evidence provided in the revised manuscript. We would be happy to work with the Reviewer to address any residual concerns.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We appreciate the reviewer’s feedback and we are pleased that our revisions have satisfied their concerns. We thank the reviewer for their helpful input during the review process to provide an improved manuscript."}]

Decision letter