Bacterial collagenase harnesses collagen geometry for processive cleavage

Domain: biology
Published: 2026-04-02
Editorial outcome: accept

Abstract
Collagen, the major structural protein in the animal extracellular matrix, forms a triple helix that resists proteolysis and requires specialised enzymes for degradation. Flesh-eating bacteria secrete collagenases that unwind the collagen triple helix and processively trim Gly–X–Y triplet repeats, yet the molecular basis of this process has remained obscure. Here, cryo-electron microscopy reveals how Hathewaya histolytica collagenase ColH engages its substrate and exploits the helix’s architecture for catalysis. ColH encircles a single collagen triple helix in a closed-ring conformation and, through dynamic domain motions, dehydrates and destabilises it. The enzyme undergoes substrate-assisted twisting to adopt a rigid ratcheted conformation, in which one chain is bent into a tripeptide-long ‘bight’ and threaded into the active site for cleavage, while two uncut strands are partitioned to non-catalytic sites. Release of the bight appears to reset the enzyme, with the uncut strands serving as guiding tracks. Repeated cycling between dynamic and rigid states likely enables triplet-by-triplet translocation, allowing ColH to harness collagen’s geometry for processive degradation. These findings reveal a bacterial strategy for collagen unwinding and cleavage distinct from that of mammalian collagenases, highlighting divergent evolutionary solutions for degrading one of nature’s most intractable substrates.

Introduction
Collagens are the primary structural components of the extracellular matrix (ECM) in animals, where they assemble into fibrillar and laminar networks that confer mechanical strength and maintain tissue organisation1, 2. Collagens possess a rod-shaped triple-helical domain, which forms a right-handed supercoil of three polypeptide chains—designated leading, middle, and trailing—staggered by one residue3. The triple-helical conformation is stabilised by repeating Gly–X–Y motifs, where, most often, X is proline, and Y is 4-hydroxyproline. This densely packed tertiary structure sterically shields peptide bonds, making collagen highly resistant to proteolysis.

To degrade collagen-based structures, bacteria and metazoa produce collagenases, i.e. zinc-dependent metallopeptidases belonging to clan MA in the MEROPS database4. Bacterial collagenases belong to the M9 family (gluzincins) based on the sequence similarity5, 6. M9A collagenases are produced mainly by marine bacteria such as species of Vibrio and Grimontia, whereas M9B collagenases are produced by terrestrial bacteria such as species of Clostridium and Bacillus. Hathewaya histolytica (formerly Clostridium histolyticum) produces two classes of M9B collagenases (ColG and ColH) 7-9 that act as virulence factors in soft-tissue infections, which have also been adapted for clinical applications, including wound debridement, fibrosis treatment, and islet isolation10. Bacterial collagenases contain multiple domains, including an activator domain (AD) and thermolysin-like peptidase domain (PD), which cooperate to unwind and cleave fibrillar collagens11. PD-mediated cleavage depends on prior AD-mediated unwinding12, 13. However, how the enzyme unwinds the triple helix and maintains the unwound state while engaging all three strands remains unclear.

Mammalian matrix metalloproteinases [MMPs, M10 family (metzincins)] have been widely studied and play a tightly regulated role in ECM remodelling. They rely on a hemopexin-like domain to assist unwinding and cleave tropocollagen at a single site via the catalytic domain14-16, with activity enhanced under tensile force17. Conversely, tensile force inhibits M9 collagenase activity18. These opposing responses suggest that M9 and M10 enzymes use fundamentally different unwinding mechanisms11-13, 15, 16, though structural insights into their molecular basis remain limited.

Unlike MMPs, which act exclusively as endopeptidases, bacterial collagenases exhibit both endopeptidase and carboxytripeptidase activities, preferentially cleaving Y–Gly bonds12, 19-22. The carboxytripeptidase processively trims Gly–X–Y triplets from the initial site of engagement towards the N-terminus. High-speed atomic force microscopy has demonstrated catalysis-coupled movement of a clostridial collagenase in the N-terminal direction23, yet how strand engagement supports this processive advancement remain unclear.

Although previous studies reported only the ‘wide-open’ conformation of M9 collagenases11, 12, 21, 24, in which the catalytically important AD and PD are spatially separated, this study presents the atomic structures of H. histolytica ColH in the ‘closed’ and ‘ratcheted’ states bound to triple-helical peptides. These structures reveal how the AD and PD cooperate to unlay, cleave, and translocate along the triple helix, defining a distinct bacterial mechanism for processive collagenolysis.

Results
ColH adopts ‘closed’ conformation
To elucidate the structural mechanisms underlying bacterial collagenase function, we performed cryo-electron microscopy (cryo-EM) on H. histolytica wild-type ColH (ColHWT; Fig. 1a–d, Supplementary Figs. 1 and 2, and Supplementary Table 1). The 2D class averages showed that ColHWT adopts a ring-like conformation of its collagenase module (CM), comprising AD and PD (Fig. 1b). The consensus cryo-EM density map, resolved at an average resolution of 2.3 Å, was well-defined for the PD but showed conformational heterogeneity in the AD (Fig. 1c). Focused refinement improved AD map quality, revealing a rigid structure with orientational flexibility relative to the other domains (Supplementary Fig. 1). Our cryo-EM analysis revealed a clear density for polycystic kidney disease-like domain 1 (PKD1) but not PKD2 or the collagen-binding domain (CBD), likely owing to linker flexibility that averages out their densities in the EM reconstruction.

The composite EM map of the AD and PD-PKD1 regions revealed a ‘closed’ CM conformation (Fig. 1d and Supplementary Figs. 1 and 2). At the junction between AD and PD, three helices (α17, α18, and α21) formed a hinge via hydrophobic interactions, whereas gate-region interactions on the opposite side reinforced the ring (Fig. 1d). PKD1 interacts with the PD in a calcium-dependent manner (Fig. 1d). A previous small-angle X-ray scattering experiment showed that calcium depletion produces an elongated apo ColH envelope25. Thus, the rigid PKD1 conformation may stabilise the overall structure and restrict PKD2-CBD orientation. We also performed X-ray crystallography of ColHWT CM at room temperature, which yielded a 2.70 Å structure essentially identical to the cryo-EM model, with a root-mean-square deviation of 1.9 Å for Cα atoms (Supplementary Fig. 3 and Supplementary Table 2). Notably, crystallographic B-factors increase progressively from the C-terminus to the N-terminus of the AD, suggesting hinge-mediated flexibility between the AD and PD (Supplementary Fig. 4).

These structural analyses suggest that ColHWT CM can accommodate a single triple-helical molecule (~15 Å diameter) in its central cavity, but gate interactions require domain opening for substrate access. To assess CM flexibility, the cryo-EM data were used for 3D variability analysis (3DVA)26, classifying particles along principal component axes. ColHWT adopts a dynamic ‘closed’ state in which CM domains undergo partial opening and twisting (Fig. 1e, f, Supplementary Fig. 5, and Supplementary Movies 1 and 2). Although low in amplitude, these motions generate an entrance of ~15 Å, sufficient to capture a tropocollagen molecule in the central cavity (Supplementary Fig. 6).

At the catalytic centre, a Zn²⁺ ion was coordinated by the conserved residues, His415, His419, and the bidentate Glu447, as well as two water molecules (w1 and w2), as observed consistently in both the cryo-EM and X-ray crystal structures, adopting hexacoordinate geometry (Supplementary Fig. 7). Neither water molecule was positioned to be deprotonated by Glu416.

ColH targets triple helix at C-terminus
To determine the substrate recognition and unwinding mechanisms, cryo-EM was performed on ColH complexed with the collagenous triple-helical peptide [(Pro-Hyp-Gly)₁₀]₃ [or (POG)₁₀] (Fig. 2, Supplementary Figs. 8, 9, 10, and Supplementary Table 1). Here, Hyp (or O) represents 4-hydroxyproline. (POG)₁₀ adopts a stable triple helix in solution (Tm = 60 °C)27. Since ColH hydrolyses (POG)₁₀ (Supplementary Fig. 11), we used a protease-null ColH mutant containing the Glu416Gln (E416Q) substitution (ColHMT).

Image analysis showed that ~80% of particles formed a 1:1 complex, with a rod-like (POG)₁₀ density protruding from one ColHMT face (Fig. 2a and Supplementary Fig. 8), whereas the remainder formed a 2:1 complex. The 1:1 complex was resolved at 2.7 Å without conformational heterogeneity except in the PKD2–CBD region. ColHMT selectively interacted with the C-terminal region of (POG)₁₀, unwinding its terminal three triplets (residues 22–30). Recognition was unidirectional, with N-to-C binding of the unwound region from the front to the back face of ColHMT (Fig. 2b, c). Neither reverse-direction recognition nor recognition involving unwinding of the central region or N-terminal side of (POG)₁₀ was detected in the cryo-EM analysis. Assuming that ColHMT can topologically entrap any site of (POG)₁₀ within its CM ring, this was not initially predicted. A previous study indicated that unwinding by M9B collagenase tends to be a local process13; therefore, ColHMT likely recognises a readily or sufficiently unwound region during N-to-C translocation, adopts a stable conformation through unidirectional binding at that site, and subsequently becomes arrested.

The N-terminal region (residues 1–15) of (POG)₁₀ remained triple-helical but was visible only at low contour levels (Supplementary Fig. 8). A triple-helix model was therefore built only for residues 16–21, where density was well defined (Fig. 2c). While ColHMT retains a ring-like conformation, resulting in only localised unwinding, structural comparison with ColHWT showed that the AD undergoes a pronounced twist of ~10 Å relative to the PD and is conformationally locked (Fig. 2d). Substrate binding thus shifts ColH toward a distorted rigid ‘ratcheted’ state, distinct from the dynamic ‘closed’ state.

Partial unfolding at the C-terminus allows the leading and middle strands of (POG)₁₀ to bind to two AD exosites (ExoLead and ExoMiddle; Fig. 2b, e). Both sites engage two contiguous triplets (Pro25–…–Gly30) via hydrophobic clefts. Proline residues at X positions (Pro25 and Pro28) form contacts with aromatic residues—Phe37, Tyr87, Phe90, and Tyr184 at ExoLead; Phe176, Trp228, and Tyr235 at ExoMiddle—through hydrophobic and CH–π interactions (Fig. 2e). Alanine substitution of these residues significantly reduced type I collagen hydrolysis but not 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-peptide) hydrolysis, highlighting their role in triple-helix recognition and unwinding (Fig. 2f). Mutations in the ExoLead residues caused a substantially greater reduction in collagenolytic activity than those in ExoMiddle (~90% versus ~60%), suggesting that ExoLead plays a more critical role in collagen recognition and unwinding. Sequence and structural comparisons with M9A and M9B collagenases showed that ExoLead residues are strictly or highly conserved across these enzymes (Supplementary Figs. 12 and 13). Consistently, their importance for collagenolytic activity in both subfamilies has been confirmed in previous mutational studies12, 13. In contrast, the aromatic residues comprising ExoMiddle in ColH are intriguingly not as well conserved within the M9 family. In the M9B subfamily, although Y235 and W228 are conserved, F176 is not, whereas in M9A, only the residue equivalent to W228 is conserved. This relatively low level of conservation may underlie the variability in substrate binding at this exosite.

Besides the aromatic interactions, several hydrogen-bonding interactions were observed (Fig. 2e). At ExoLead, such interactions stabilise the leading-strand conformation: the main-chain carbonyls of Hyp26 and Gly27 hydrogen bond with Arg83, whereas the OD1 atom of Hyp29 forms hydrogen bonds with Glu80 and Arg83. Although hydrogen-bonding networks at ExoMiddle are less extensive, an interaction between the carbonyl group of Hyp26 and Asn232 as well as OD1 of Hyp29 and carbonyl group of Asp224 stabilises the middle strand. While these interactions further strengthen peptide binding, the residues are not strictly conserved within the M9 family, with the exception of Arg83 (Supplementary Figs. 12 and 13). The importance of arginine at this position for substrate recognition in M9A and M9B collagenases has been demonstrated in mutational studies12, 13, consistent with our structural observation that Arg83 engages and stabilises the main chain of the leading strand.

Most Hyp residues remained solvent-exposed and contributed little to binding, consistent with the cryo-EM data of ColHMT bound to [(Pro–Pro–Gly)₁₀]₃ [or (PPG)₁₀] (Supplementary Fig. 14 and Supplementary Table 1), which revealed a binding mode similar to that of the ColHMT–(POG)₁₀ complex (Supplementary Fig. 15). These results suggest that ColH has evolved to recognise Gly–Pro–Yaa–Gly–Pro–Yaa motifs in native collagen via its hydrophobic exosites, consistent with previous proteomics analyses demonstrating the substrate specificity of clostridial collagenases for Gly–Pro–Yaa triplets28.

Although the leading and middle strands followed a triple-helical axis with minor deviation, the trailing strand veered sharply, threading the three triplets at its C-terminus (Gly21–…–Gly30; P6 to P4′) into the active-site cleft (Fig. 2c, e). This distortion is critical for Zn²⁺ coordination of the scissile carbonyl oxygen. As observed in the inhibitor-bound structures of other M9B collagenases11, 24, the active-site cleft embraced by the N- and C-terminal half-domains of the PD catalytic subdomain was contracted as much as 3 Å, holding the trailing strand tightly (Fig. 2d and Supplementary Fig. 16). In the cleft, the Hyp26 (P1)–Gly27 (P1′) scissile motif was stabilised by antiparallel interactions with conserved Gly385 and Gly386 on the β4 edge strand, known as the double-glycine S1′ recognition site11, 24. Ser405 of the ‘wall motif’24 engaged the Hyp29 (P3′) carbonyl, acting as a molecular ruler for substrate recognition. On the opposite side, Trp431 interacted with Pro25 (P2) and the Cα–H of Gly24 (P3) through CH–π interactions and, together with Tyr491, interacted with Pro22 (P5) via nonpolar contacts and shape complementarity, thereby stabilising the three-residue bight (Fig. 2g). Notably, Trp431 lies on a calcium-binding loop located between the N- and C-terminal half-domains, where a conserved glutamate side-chain (Glu390 in ColH) anchors the edge strand and calcium, thereby bracing the two half-domains (Fig. 2g). Consequently, calcium binding not only stabilises zinc coordination in the active site but also ensures the proper positioning of Trp431. These observations support the essential role of Ca²⁺ coordination in ColH catalysis24.

When viewed from the front face of ColHMT, three aromatic residues, Phe90, Tyr235, and Trp431, engaged the interacting triplets as aromatic pivots marking the transition from a helical to unwound structure (Fig. 2h). These residues may promote local bending to promote strand unwinding or favour partially unfolded triple-helical structures, facilitating recognition of the frayed C-terminal region of (POG)₁₀29. Substitution of Phe90 greatly reduced collagenolytic activity, whereas Tyr235 mutation led to a more modest decrease (~60% versus ~30%; Fig. 2f). Although Tyr235 is not conserved in the M9A family (Supplementary Fig. 12), a phenylalanine residue that may serve a similar function is positioned nearby (Supplementary Fig. 13). This suggests that an aromatic residue at this position supports the binding and stabilisation of the unwound collagen triple helix.

Strikingly, the substitution of Trp431 with alanine resulted in a dramatic decrease (~95%) in collagenolytic activity and almost complete loss of enzymatic activity toward Pz-peptide, comparable to that observed for the catalytically inactive E416Q mutant (Fig. 2f). Circular dichroism (CD) measurements confirmed comparable structures between the mutants and ColHWT (Supplementary Fig. 17). Trp431 lies near the catalytic centre (within 10 Å from zinc to the indole ring) via the calcium-binding loop, and its indole ring stabilises Pro25 (P2) via a CH–π interaction (Fig. 2g). A structural model of ColH with Pz-peptide based on the ColHMT–(POG)₁₀ complex indicated that this interaction is also responsible for the loss of Pz-peptidase activity, as the Pro1 residue of Pz-peptide is predicted to be in the S2 subsite and interacts with Trp431 (Supplementary Fig. 18). These results further substantiate the critical role of Trp431 in ensuring stable substrate binding and correct orientation within the active-site cleft for efficient hydrolysis.

At the catalytic centre, the scissile peptide carbonyl oxygen coordinated the Zn²⁺ ion, increasing polarisation of the carbonyl bond and priming it for nucleophilic attack. No ordered water molecules were observed near either the scissile bond or residue Gln416. The carbonyl oxygen lies too close (3.2 Å) to the amide oxygen of Gln416, resulting in a non-productive pentacoordinate geometry and complete loss of hydrolytic activity (Fig. 2f). Since the corresponding distance in a wild-type structure of Grimontia collagenase Ghcol in complex with (GPO)₂ is 4.6 Å30, enough to evoke a substrate water molecule to the space, Glu416 in ColHWT may deprotonate a water molecule to initiate a nucleophilic attack.

ColH recognises triple-helical substrate
During image processing, we observed particles of a 2:1 complex (Fig. 2a). 3D reconstruction revealed one ColHMT molecule bound to the (POG)₁₀ C-terminus (ColHMT-C) and another to the N-terminus (Fig. 3a and Supplementary Fig. 19). Consensus maps and 3DVA revealed ColHMT-N to be more dynamic than ColHMT-C, resulting in a lower resolution for ColHMT-N (Supplementary Fig. 10 and Supplementary Movie 3). In contrast, (POG)₁₀ density was well resolved and unexpectedly revealed a one-triplet mis-stagger in the leading strand (Fig. 3a, b). Although such mis-staggered helices are rare31, they may have facilitated additional ColHMT recruitment under our experimental conditions.

Local refinement improved the resolution to 3.4 Å for ColHMT-N and 3.1 Å for ColHMT-C, enabling model building (Fig. 3c and Supplementary Fig. 9). In the 2:1 complex, ColHMT-N interacted with the leading strand via ExoLead of the AD without major unwinding, while ColHMT-C unwound the mis-staggered C-terminus similarly to the 1:1 complex (Fig. 3c, d). These results highlight the critical role of ExoLead in triple-helix recognition. As with ColHMT-C, the hydrophobic residues Phe37, Tyr87, and Phe90 mediated ExoLead binding. Although the protruding N-terminal triplet may aid initial recognition, ColHMT interacted more specifically with the Pro4–Hyp5–Gly6–Pro7–Hyp8–Gly9 segment (Fig. 3d). The N-terminal triplet adopted a bent conformation, likely due to steric clashes with aromatic residues (Tyr87, Phe90, and Tyr184). We also observed a slight displacement of the middle chain (Pro1–Hyp2) toward ExoMiddle. These results suggest that ExoLead aromatic residues promote local destabilisation of the triple helix during ColH translocation from the N- to the C-terminus of tropocollagen. This interpretation is supported by the extended (POG)n model, which shows steric clashes at ExoLead (Fig. 3e). Nevertheless, ExoLead-driven unwinding of (POG)₁₀ in the complex may be restricted by steric hindrance between the PKD1 of ColHMT-N and CM of ColHMT-C already bound at the C-terminus (Fig. 3a and Supplementary Fig. 19). This steric hindrance could impede C-terminal progression of ColHMT-N and thereby limit further N-terminal unravelling of (POG)₁₀, potentially reducing stable engagement of the partially unwound middle and trailing strands by ExoMiddle and the active site, respectively.

ColH unwinds triple-helical substrate
To improve the resolution of the 2:1 complex, we employed a longer peptide, [(Pro–Hyp–Gly)₁₂]₃ [or (POG)₁₂] (Fig. 4a, Supplementary Fig. 20, and Supplementary Table 1). Unlike for (POG)₁₀, 2D class averages of the ColHMT–(POG)₁₂ complex showed only a 1:1 assembly (Fig. 4b), yielding an unanticipated but informative structure. The 3D reconstruction (2.8 Å resolution) resembled ColHWT in the ‘closed’ state, with the AD displaying conformational heterogeneity rather than the rigid ‘ratcheted’ conformation (Supplementary Fig. 20). Local refinement improved AD and PD–PKD1 resolutions to 2.9 and 2.8 Å, respectively. Residual EM density for (POG)₁₂ was observed at three positions: Gly–Pro–Hyp–Gly–Pro–Hyp–Gly motifs at ExoLead and ExoMiddle and a longer Gly–Pro–Hyp–Gly–Pro–Hyp–Gly–Pro–Hyp–Gly stretch at the active site (Fig. 4c–e). These binding modes mirrored those in the 1:1 ColHMT-C–(POG)₁₀ complex (Figs. 2e and 4e). Even at low contour levels, no triple-helical density was detected, indicating complete unwinding of (POG)₁₂ by ColHMT.

We initially hypothesised that one ColHMT molecule would unwind the three C-terminal triplets of (POG)₁₂, allowing a second ColHMT to bind to the N-terminus—either capping it or unwinding the first three triplets—leaving the central six triplets as a stable helix, since (Pro–Hyp–Gly)₆ forms a helix at room temperature (Fig. 4f)32. However, this was inconsistent with the 2D class averages (Fig. 4b and Supplementary Fig. 20). Instead, cryo-EM revealed complete unfolding of (POG)₁₂, suggesting that the additional ColHMT binding locally promotes progressive destabilisation or unwinding in an N-to-C direction, likely in a spiral manner, approaching the bound ColHMT, until a steric hindrance occurs due to its PKD1. The remaining central segment, now reduced to 3–4 triplets, appears insufficient to maintain a triple-helical structure (Fig. 4f). Here, we cannot exclude that a second ColHMT binds adjacent to ColHMT-C and subsequently migrates toward the N-terminus, inducing global unwinding of (POG)₁₂. Once fully unwound, the two molecules are difficult to analyse collectively owing to their orientational flexibility or possible dissociation of one molecule from the unwound peptides.

Since the CM in the ColHMT–(POG)₁₂ complex shows a dynamic ‘closed’ state, these findings indicate that ColH can adopt the ‘ratcheted’ state only when the collagen substrate remains partially unwound, with the N-terminal side retaining its triple-helical structure. These results suggest that ColH exploits the intrinsic architecture of the triple helix to achieve the ratcheted state—a transient conformation that mediates strand unlaying, strand-specific cleavage, and N-terminal-directed translocation, as discussed in the following section.

Methods
Plasmids
Escherichia coli DH5α was used for plasmid construction. To express ColHWT, the plasmid pGEX-ColH was generated by amplifying the coding sequence of mature ColH (Val1–Arg981) using the primer pair 5′-ggatccGTACAAAATGAAAGTAAGAGGTATAC-3′ and 5′-ctcgagTTATCTTCCTACTGAACCTTCTATAT-3′ (lowercase letters indicate BamHI and XhoI recognition sites, respectively), followed by insertion into pGEX-4T-2 (Cytiva) at the BamHI and XhoI sites. Mutant ColH plasmids were generated by site-directed mutagenesis using a PrimeSTAR Mutagenesis Basal Kit (TaKaRa Bio) with the primers listed in Supplementary Table 3. To express the ColHWT collagenase module (CM; Val1–Ser686), pGEX-ColHCM was constructed by PCR amplification with primers 5′-cgcggatccGTACAAAATGAAAGTAAGAGGTATACA-3′ and 5′-ccgctcgagTTATGAATTTTTGGAATCACCTTCGTTTGG-3′ (lowercase letters indicate flanking trinucleotides followed by BamHI and XhoI sites, respectively) and inserted into pGEX-4T-2. All constructs were verified by using Sanger sequencing.

Purification of wild-type and mutant ColH
E. coli BL21-CodonPlus-RIL (Agilent Technologies) cells transformed with plasmids encoding ColHWT or mutants were grown in 2YT medium (1.6% tryptone, 1.0% yeast extract, 0.5% NaCl) containing 2% glucose, 50 µg/mL ampicillin, and 30 µg/mL chloramphenicol at 37 °C until reaching an OD600 of 1.0. Cultures were cooled in an ice bath for 10 min, and expression was induced with 1 mM isopropyl-β-D-thiogalactopyranoside at 25 °C for 20 h. Cells were harvested by centrifugation at 7500 g and 4 °C for 10 min and resuspended in phosphate-buffered saline (PBS) with 1 mM phenylmethylsulfonyl fluoride. Cells were disrupted using a French press at 10 000 psi, treated with Triton X-100 (1% final concentration), and the lysate was rotated at 4 °C for 30 min before clarification by centrifugation at 25 000 g and 4 °C for 30 min, repeated twice. The cleared lysate was incubated with Glutathione Sepharose 4 Fast Flow beads (Cytiva) at 4 °C for 30 min, washed with PBS, and transferred to a column. GST-fusion proteins were eluted with 50 mM Tris-HCl (pH 8.0) containing 10 mM glutathione, and peak fractions were pooled. Thrombin protease (Cytiva) was added to remove the GST tag, and the sample was incubated at 25 °C for 14 h. The digest was dialysed four times against 50 mM Tris-HCl (pH 7.5) containing 1 mM CaCl₂, applied to a Glutathione Sepharose column, and flow-through fractions containing ColH were collected. For structural studies, fractions were concentrated by ultrafiltration with Amicon Ultra 30K centrifugal filters (Millipore) and loaded onto a HiPrep Sephacryl S-200HR 16/60 column (Cytiva) pre-equilibrated with 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1 mM CaCl₂.

Purification of ColHWT collagenase module
Purification of the ColHWT CM followed the same procedure as for ColHWT, except that protein expression was induced at 30 °C for 6 h. After removal of the GST tag by affinity chromatography, proteins were purified by ion-exchange chromatography with Q-Sepharose Fast Flow beads (Cytiva) equilibrated with 50 mM Tris-HCl (pH 7.5) containing 1 mM CaCl₂. The flow-through fraction containing ColHWT CM was further purified by size-exclusion chromatography as described above.

Cryo-EM sample preparation and data collection
Purified ColHWT or mutant was diluted to 1.0 mg/mL in cryo-EM buffer (50 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1 mM CaCl₂). ColHMT was mixed with the synthetic peptide (POG)₁₀, (PPG)₁₀, or (POG)₁₂ at a 1:2 molar ratio, adjusted to a final protein concentration of 1.0 mg/mL, and incubated on ice for 30 min. (POG)₁₀ and (PPG)₁₀ were purchased from Peptide Institute (Osaka, Japan), and (POG)₁₂ from Bio-Synthesis (Lewisville, TX, USA), and were dissolved in cryo-EM buffer. Peptide solutions were heated at 95 °C for 5 min, cooled to room temperature for 10 min, and incubated at 4 °C for ≥24 h for triple-helix formation. Each sample was applied to glow-discharged copper grids (R1.2/1.3 200 mesh; Quantifoil, Großlöbichau, Germany) for 10 s at 10 mA using a JEC-3000FC Auto Fine Coater (JEOL). Grids were blotted for 3 s at 4 °C (100% humidity) with a blot force of 0 and plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, USA). Screening and data collection were performed on CRYO ARM 300 and CRYO ARM 300 II microscopes (JEOL) operating at 300 kV and equipped with a cold field-emission gun and K3 direct electron detector (Gatan, Pleasanton, CA, USA). Cryo-EM movies were recorded using SerialEM (version 4.1.6)38 with 5 × 5 beam-image shift patterns (coma vs. image shift calibrated before acquisition). The data collection parameters are listed in Supplementary Table 1.

Cryo-EM data processing
All data processing was performed in CryoSPARC (version 4.4.1-4.6.0)39. Imported movies were subjected to beam-induced motion correction using Patch Motion Correction, and the contrast transfer function (CTF) parameters were estimated using Patch CTF estimation.

For ColHWT, 16,098,214 particles were automatically picked using Template Picker with templates generated from the ColHWT CM crystal structure (PDB ID: 9CME [http://doi.org/10.2210/pdb9CME/pdb]) and extracted from 8,150 micrographs with a box size of 128 pixels (1.86 Å/pixel). After 2D classification to remove junk particles, the selected particles were subjected to several rounds of ab initio reconstruction and heterogeneous refinement. A total of 3,488,844 good-quality particles were re-extracted with a box size of 256 pixels at 0.87 Å/pixel, further classified, and refined to obtain 1,533,374 particles. Further homogeneous refinement, non-uniform refinement, and reference-based motion correction yielded 1,401,659 high-quality particles and a 2.3 Å consensus map. 3DVA26 was performed at 3 Å (Supplementary Fig. 5 and Supplementary Movies 1 and 2). Focused refinement of the AD and PD-PKD1 regions improved map quality. A composite map was generated using phenix.combine_focused_maps in PHENIX (version 1.19.2-4158 or 1.20.1-4487)40.

For the 1:1 ColHMT–(POG)₁₀ complex [ColHMT bound to the C-terminal region of (POG)₁₀], 13,017,170 particles were picked with templates generated from the ColHWT map and extracted from 7,349 micrographs with a box size of 120 pixels (1.74 Å/pixel). The extracted particles were subjected to several rounds of 2D classification, ab initio reconstruction, and heterogeneous refinement to filter junk particles. A total of 3,551,012 good-quality particles were re-extracted with a box size of 240 pixels (0.87 Å/pixel) and subjected to further rounds of 2D classification, ab-initio reconstruction, and heterogeneous refinement. After homogeneous refinement and subsequent non-uniform refinement, 534,057 particles were motion-corrected using reference-based motion correction to yield a cryo-EM map of 2.6 Å resolution. To exclude particles in which two ColHMT molecules were bound to (POG)₁₀, 503,128 particles were re-extracted with a box size of 400 pixels (0.87 Å/pixel) and subjected to 2D classification. A further 3D classification yielded three classes with ColHMT bound to (POG)₁₀ with a 1:1 stoichiometry and one class with bound ColHMT to (POG)₁₀ with a 2:1 stoichiometry. From the classes with only one ColHMT bound, a total of 396,749 particles were re-extracted with a box size of 240 pixels (0.87 Å/pixel) and subjected to homogeneous refinement, non-uniform refinement, and reference-based motion correction. After homogeneous and non-uniform refinement, local refinement using 369,742 particles yielded a cryo-EM map of ColHMT bound to the C-terminal region of (POG)₁₀ at a 2.7 Å resolution.

For the 2:1 ColHMT–(POG)₁₀ complex, 6,141,403 particles were picked with templates generated from the ColHWT map, extracted from 7,385 micrographs with a box size of 120 pixels (2.32 Å/pixel), and subjected to several rounds of ab initio reconstruction and heterogeneous refinement to filter junk particles. A total of 745,629 good-quality particles were re-extracted with a box size of 300 pixels (0.87 Å/pixel) and subjected to several rounds of ab initio reconstruction and heterogeneous refinement. After homogeneous refinement and subsequent non-uniform refinement, 58,438 particles were subjected to reference-based motion correction. After homogeneous refinement, non-uniform refinement yielded a consensus map of the 2:1 ColHMT–(POG)₁₀ complex at a 3.3 Å resolution. Using these particles, 3DVA26 was performed with a filter resolution of 5 Å (Supplementary Fig. 10, and Supplementary Movie 3). The quality of the map was improved by focused refinement of ColHMT bound to the C- and N-terminal regions of (POG)₁₀. A composite map of the 2:1 ColHMT–(POG)₁₀ complex was generated using phenix.combine_focused_maps in PHENIX40.

For the ColHMT–(PPG)₁₀ complex, 14,102,948 particles were automatically picked with templates generated from the ColHWT cryo-EM map and extracted from 8,743 micrographs with a box size of 120 pixels (1.74 Å/pixel). The extracted particles were subjected to several rounds of 2D classification, ab initio reconstruction, and heterogeneous refinement to filter junk particles. A total of 3,773,237 good-quality particles were re-extracted with a box size of 240 pixels (0.87 Å/pixel) and subjected to several rounds of ab initio reconstruction and heterogeneous refinement. After homogeneous refinement and subsequent non-uniform refinement, 570,503 particles were subjected to reference-based motion correction. After homogeneous refinement and non-uniform refinement, local refinement using 569,950 particles yielded a cryo-EM map of the 1:1 ColHMT–(PPG)₁₀ complex with a 2.2 Å resolution.

For the ColHMT–(POG)₁₂ complex, 10,114,389 particles were automatically picked using Template Picker with templates generated from the cryo-EM map of ColHMT bound to the C-terminal of (POG)₁₀ and extracted from 6,875 micrographs with a box size of 120 pixels (1.72 Å/pixel). The extracted particles were subjected to several rounds of ab initio reconstruction, heterogeneous refinement, and 2D classification to filter junk particles. A total of 2,069,705 good-quality particles were re-extracted with a box size of 240 pixels (0.86 Å/pixel) and subjected to several rounds of ab initio reconstruction and heterogeneous refinement. Further 3D classification yielded one class with a clear map for the AD ExoLead and ExoMiddle regions. After homogeneous and non-uniform refinement, 318,280 particles were subjected to reference-based motion correction. After homogeneous refinement, non-uniform refinement yielded a consensus map of the ColHMT–(POG)₁₂ complex at a 2.8 Å resolution. The quality of the map was improved by focused refinement of the AD and PD-PKD1 regions. A composite map of the ColHMT–(POG)₁₂ complex was generated using phenix.combine_focused_maps in PHENIX40.

The global resolution of each cryo‑EM map was estimated using the gold‑standard Fourier shell correlation (FSC) between independently refined half‑maps with the 0.143 criterion. The resolution of the atomic model was defined as that at the FSC = 0.5 cutoff in the model‑to‑map FSC curve. For composite maps, the composite half‑maps produced using phenix.combine_focused_maps were imported into CryoSPARC39 and processed with the Validation (FSC) job to generate gold‑standard map‑to‑map FSC curves. The global resolution of each composite map was defined at the FSC = 0.143 threshold. These model-to-map and map-to-map FSC curves and corresponding resolution values are summarised in Supplementary Table 1 and shown in Supplementary Figs. 1, 8, 9, 14, and 20.

Cryo-EM model building and refinement
For ColHWT, an initial model was built with ModelAngelo41 based on the ColH amino acid sequence, manually refined in Coot (version 0.8.9.2 or 0.9.8.93)42, and subjected to real-space refinement using phenix.real_space_refine in PHENIX40. Water molecules were automatically placed in Coot42 and manually verified. The final model comprised residues Gln2–Asp770.

For the 1:1 ColHMT–(POG)₁₀ and 1:1 ColHMT–(PPG)₁₀ complexes, initial models were built in ModelAngelo41 with sequences of ColH and the respective peptides. Manual refinement was performed in Coot42 and real-space refinement in PHENIX (using phenix.real_space_refine) 40. Water molecules were automatically located using Coot42 and manually verified. Each final model contained three collagen peptide segments (Pro16–Gly30 of the leading, middle, and trailing chains) bound to ColHMT.

For the 2:1 ColHMT–(POG)₁₀ complex, initial models were generated by superimposing the 1:1 ColHMT–(POG)₁₀ and ColHWT structures onto the composite map in ChimeraX (version 1.9-1.10.1)43. After manual and real-space refinements, the final model was generated with one ColHMT bound to each (POG)₁₀ terminus.

For the ColHMT–(POG)₁₂ complex, the 1:1 ColHMT–(POG)₁₀ structure was fit into the map in ChimeraX43. Non-visible peptide regions were removed, followed by refinement in Coot42 and PHENIX40 (using phenix.real_space_refine). The final model included two (POG)₁₂ peptide segments (Gly-Pro-Hyp-Gly-Pro-Hyp-Gly) bound to ExoLead and ExoMiddle, respectively, and one additional segment (Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly) at the active site.

All structures were validated with MolProbity44 in PHENIX40. The refinement statistics are provided in Supplementary Table 1.

Crystallisation
High-throughput screening using a Hampton Research HR2-144 index on an Oryx 8 (Douglas Instruments) identified several crystallisation conditions. ColHWT CM at 7.7 mg/mL in 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 1 mM CaCl₂ produced crystals within 24–48 h. Optimal conditions were 0.1 M Bis-Tris (pH 6.5), 0.2 M magnesium chloride hexahydrate, and 25% (w/v) polyethylene glycol (PEG) 3350, supplemented with Izit Crystal Dye (Hampton Research) for visualisation. Microseeds were prepared following the protocol of D’Arcy et al.45, adapted from Luft and DeTitta46. Briefly, 20–50 µL of reservoir solution was chilled on ice in a seed bead tube, and the crystals were crushed for 30 s and rinsed in cold reservoir solution; this process was repeated until no visible crystals remained. The seed stock was vortexed four times for 30 s and stored at –80 °C.

X-ray crystallography and structure analysis
Tetragonal crystals grown using the sitting-drop method with microseeding were mounted in sealed quartz capillaries. Diffraction data were collected at room temperature on a Rigaku XtaLAB Synergy-S equipped with a copper sealed-tube X-ray source and HyPix600HE detector. A crystal from a reservoir containing 1.0 M MgCl₂·6H₂O, 1.0 M Tris-HCl (pH 8.5), and 50% (w/v) PEG 3350 was selected. Data were processed using CrysAlisPro (Rigaku Oxford Diffraction) and Aimless from the CCP4 suite (version 8.0.019)47. Phases were determined by molecular replacement using Phaser in PHENIX40 with the ColH PD segment (PDB ID: 4AR1 [http://doi.org/10.2210/pdb4AR1/pdb]) and ColG AD (PDB ID: 4ARE [http://doi.org/10.2210/pdb4ARE/pdb]) as search models. Iterative rounds of manual rebuilding were performed in Coot42, real-space refinement in PHENIX40, and reciprocal-space refinement with twin correction (–h, l, k) in REFMAC5 from the CCP4 suite47. The refined structure showed 95.11% of residues in favoured regions and 4.89% in allowed regions and no Ramachandran outliers. The data collection and refinement statistics are summarised in Supplementary Table 2.

Enzyme assays
Protein concentrations were determined with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) using bovine serum albumin as a standard. Hydrolytic activity toward a non-helical collagen peptide was measured with Pz-peptide (Sigma-Aldrich, St. Louis, MO, USA) as described by Wünsch and Heidrich48. Collagenolytic activity against insoluble bovine Achilles tendon (code CL; Worthington Biochemical, Freehold, NJ, USA) was measured following the manufacturer’s instructions. Briefly, 25 mg of collagen was swollen in 5 mL of 50 mM Tris(hydroxymethyl)-methyl-2-aminoethane sulfonate and 0.36 mM CaCl₂ (pH 7.5) for 30 min at 37 °C. Reactions were initiated by adding 0.1 mL enzyme solution containing 10 µg protein and incubated for 5 h at 37 °C. The supernatant was collected by centrifugation at 4 °C, and liberated amino acids were quantified using ninhydrin assays, expressed as leucine equivalents. All assays were performed in triplicate across three independent experiments. To compare the statistical differences between wild-type and mutant ColH, a two-tailed t-test was used (Fig. 2f).

Digestion assays of triple-helical peptides
(POG)₁₀ and (PPG)₁₀ peptides were dissolved in water (1 mg/mL), heated at 95 °C for 5 min, cooled to room temperature for 10 min, and incubated at 4 °C for ≥24 h to allow triple-helix formation. Digestion reactions with ColHWT were performed in 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1 mM CaCl₂. (POG)₁₀ (0.1 mg/mL) was incubated with ColHWT (0.01 mg/mL) at 4 °C for 1, 9, or 24 h, or at 37 °C for 1 or 3 h. At a higher enzyme concentration (0.1 mg/mL), reactions were conducted at 4 °C for 1, 3, or 24 h. For (PPG)₁₀ (0.1 mg/mL), digestion using 0.01 mg/mL ColHWT was conducted at 4 °C for 1 or 2 h.

At each time point, aliquots were analysed by reversed-phase high-performance liquid chromatography on a COSMOSIL 5C18-AR-II column (4.6 × 250 mm; Nacalai Tesque, Kyoto, Japan) at 60 °C, with UV detection at 220 nm. Separations were run at 1 mL/min with linear acetonitrile gradients (10–40% or 0–30% over 30 min) in water, both containing 0.05% trifluoroacetic acid. Peaks corresponding to intact peptides were integrated, and the percentage of residual substrate was calculated relative to the undigested controls.

Sequence alignment
Amino-acid sequences from three previously determined structures and one newly determined structure—two M9A collagenases (Ghcol21 and VhaC12) and two M9B collagenases (ColG11 and ColH)—were aligned using Clustal Omega (version 1.2.4)49. Structural alignment of their AD and catalytic subdomain was performed in Coot42 to identify structurally equivalent residues and refine the sequence alignment. Secondary-structure elements for ColH were assigned using DSSP (version 4)50.

Circular dichroism (CD) spectroscopy
Far-UV CD spectra were recorded using a Jasco J-720 spectropolarimeter (JASCO, Tokyo, Japan) to assess the secondary structure of the purified proteins. Protein samples were prepared in compatible buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM CaCl₂) at a final concentration of ~0.5 mg/mL. CD measurements were performed at 25 °C using a synthetic quartz cuvette with a path length of 1 mm. Spectra were collected over a wavelength range of 200–250 nm with a bandwidth of 1 nm, a scanning speed of 200 nm/min, and a response time of 0.5 s. A buffer spectrum recorded under identical conditions was subtracted from each protein spectrum.

Structure modelling
The structures of ColHWT and Pz-peptide complexes were modelled using ColHMT-(POG)₁₀ (PDB ID: 9LQJ [http://doi.org/10.2210/pdb9LQJ/pdb]) as the initial model. In ColHMT-(POG)₁₀, three collagen-like peptides form a complex with ColH, and the two collagen-like peptides bound to the AD were removed. For the remaining collagen-like peptides bound to the PD, only the region around the active site was retained and residue Q416 was mutated back to E416. Based on the remaining collagen-like peptide, we modelled the Pz-peptide in Molecular Operating Environment (MOE) software (ver. 2020, Chemical Computing Group, Inc.)51. The protonation in ColHWT and Pz-peptide complexes was performed using Protonate 3D in MOE and optimisation considered the ligand to surrounding 4.5 Å amino acid residues with a constraint (tether = 1.0) under the AMBER10: EHT force field52, 53.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript Oki H et al. describe cryo-EM and X-ray structures of the H. histolytica collagenase ColH, a processive\ncarboxytripeptidase. The structures presented reveal distinct states of collagen triple helix engagement and unwinding,\nproviding unprecedented insight into the mechanism of collagen engagement and cleavage. Overall, this is a very well\nperformed study that sheds new light on an interesting bacterial collagenase with potential for future engineering or inhibitor\ndevelopment. For the most part the manuscript is very clearly written, and should be easily understood even by a non-expert\nin the field. There are extensive supplemental and extended data figures that are well prepared to deliver the overall story.\nOverall, I am highly supportive of publication of this manuscript, as I feel the studies have been rigorously performed with\nappropriate analysis. I have only minor comments for the authors to consider before final publication...\n1. Line 98 - \"Although low in amplitude, these motions generate an entrance of ~15Å, sufficient to capture a tropocollagen\nmolecule in the central cavity\". It may be nice to show a (supplemental/extended) figure with a tropocollagen molecule at\nscale next to the opening. It is hard to tell from the 3DVA alone just how large the opening would need to be in order for the\n\"clamp\" to open sufficiently to accommodate entry of tropocollagen.\n2. While the resolutions reported in most of the cryo-EM experiments are indeed possibly suitable for placement of water\nmolecules, I am skeptical of the placement and total number of water molecules included in the refined structures. In\nparticular, in Figure 1B and 1D it is clear that the resolution in the Activator Domain is significantly lower due to inherent\nflexibility, yet water molecules are still modelled around the periphery of the activator domain. I am skeptical that these are\ntruly water molecules, rather than simply \"dust\" generated by the automatic masking process in cryosparc. While well\nordered water molecules in the protein interior with good stable hydrogen bonding are likely observed in the best resolved\nregions, the waters that are placed at the periphery of the protein (and especially in the Activator Domain) are skeptical at\nbest. It may be worthwhile for the authors to carefully review the placement of water molecules.\n3. In addition to the point above, can the authors clearly see water molecules near the zinc active site? Including a\nsupplemental figure demonstrating well resolved water molecules around the zinc active site would be ideal. From the\nplacement of these water molecules is it possible to delineate the precise positioning of the water that will attack the peptide\ncarbbonyl? This information could be very useful to those studying zinc metalloproteases more generally.\n3. Something seems weird in the methods section about box and pixel sizes. Line 386 - \"with a box size of 256 pixels (1.86\nÅ/pix) ... particles were re-extracted with a box size of 256 pixels at 0.87 Å/pix\". I think what the authors mean to say is that\nparticles were originally extracted at a box size of ~550 pixels and downsampled to a box size of 256 pixels (1.87Å) before\ninitial rounds of 2D classification? I don't see how the particles could be extracted with the same box size and different pixel\nsizes unless the micrographs themselves were binned at some point, or during extraction the particles were also\ndownsampled. Please clarify in all sections throughout the methods.\n4. Line 118 - \"unwinding its terminal three triplets in an N-to-C direction\". This statement confused me when I first read it.\nCouldn't one argue that the actual unwinding occurs from a C-to-N direction? Figure 5 makes this a little more clear, but at\nthis point in the manuscript I was slightly confused by this wording and the diagram shown in Figure 2. Please see if revision\nmakes sense, or this statement is accurate. As a reader I may be getting confused between \"unwinding\" and \"translocation\".\n5. Line 149 - it may be good to include a reference for the role of Calcium coordination in catalysis.", "reviewer_2": "The collagenolytic mechanism of M9 collagenases has been extensively studied. The collagen triple helix is the minimal\nstructural unit of collagen, and it is generally believed that this triple helical molecule must be locally unwound to release a\nsingle peptide chain into the catalytic cavity before hydrolysis (2012 NSMB; 2022 NC; 2024 PNAS). However, direct\nevidence showing that any M9 collagenase actively drives this unwinding process has been lacking. In this context, the work\nby Oki et al. provides valuable structural and biochemical insights. The authors cleverly utilized a collagen synthetic peptide\nthat forms a stable triple helix in solution and resolved cryo-EM structures of M9B collagenase ColH in complex with POG10\nin both its triple helical and unwound forms. Combined with biochemical assays, this work explores the unwinding\nmechanism of the collagen triple helix by ColH. Despite these important contributions, there are several concerns that need\nto be addressed before publication.\n1. From a three-dimensional structural perspective, the W431 residue is far from the catalytic center of ColH. However, as\nshown in Fig. 2g, the W431 mutation to alanine not only significantly reduced the activity of collagen but also almost\ncompletely lost the activity of Pz peptide. There is no doubt that this phenomenon is counterintuitive. Although the author\nprovided relevant explanations in L148-L149, the significance of the W431 residue of ColH in triple helix unwinding or\ncatalysis deserves to be reconsidered. The solution to this question would benefit from circular dichroism spectroscopy\nanalysis of mutant W431A and even other mutants to determine that the decreased collagen-binding ability of the mutants is\ncaused by amino acid replacement rather than structural alteration. In addition, given the significance of the W431 residue in\nthe triple helix unwinding process, the authors are encouraged to resolve the complex structure of W431A and POG10.\n2. L146: What kind of interaction exists between W431 and P25 and P22? It is recommended a close-up view of the relative\npositions or interactions between relevant amino acids in Fig. 2e.\n3. In the complex structure of ColH and POG12, why is the triple helix form of POG12 not visible? Can the author make a\nreasonable speculation and elaborate on it in the text? Is it related to its own properties of POG12 or did the enzyme\nmolecule ColH mediate the complete unwinding of the triple helix molecule?\n4. The conservation of key sites (W431, F90, Y235, etc.) related to triple helix unwinding throughout the M9 family\ncollagenases (M9A and M9B) needs to be explored.\n5. The proposed mechanism is mainly inferred from structural data, lacking direct kinetic or biochemical evidence. The\ndiscussion should explicitly acknowledge this limitation.\n6. The feasibility of ColH fully wrapping around a single collagen chain within densely packed fibrils is questionable. The\nauthors should further discuss how ColH might act on native collagen substrates, going beyond the simple statement that\nColH targets only partially unwound or damaged regions of collagen fibrils. In addition, the potential auxiliary roles of PKD\nand CBD domains in substrate recognition, binding, and unwinding should be considered to strengthen the physiological\nrelevance of the proposed mechanism.", "reviewer_3": "The manuscript by Oki and colleagues entitled \"Bacterial Collagenase Harnesses Collagen Geometry for Processive\nCleavage” reports on a study that investigates the mechanism of how bacterial collagenases unwind and cleave collagen.\nCollagen’s triple-helical fold and high content of (hydroxy-)proline render the molecule extremely stable and resistant to\nenzymatic proteolysis within the context of the extracellular matrix. To overcome this difficulty, true collagenases, i.e. those\nenzymes that process natively folded triple-helical collagen, must unwind the molecule locally to make the cleavage sites\naccessible to the enzyme. Based on high-resolution cryo–electron microscopy findings the authors demonstrate that ColH\nencircles a single collagen triple helix via its AD and PD domains. Via the hydrophobic AD exosites the triple helix is\neventually stripped of its stabilizing hydration shell by the enzymes, and the substrate’s leading and middle strand are bound\nand sterically distorted by binding to the two AD exosites. The displaced trailing strand is threaded via “bight” formation\nmediated by Trp431 into the active-site cleft. Exploiting collagen’s geometry for continuous degradation, the authors propose\nthat small-amplitude AD-PD movements, i.e. cycling between “ratcheted” and “closed” stated, allow for triplet-by-triplet\ntranslocation and its stepwise processing.\nThe present work is of great importance to the field and beyond, and deserves publication in Nature Communications.\nMerely, the following minor issues should be dealt with in a revised version of the manuscript before prior to acceptance for\npublication.\nThe numbering of the references is not chronological. Please correct.\nIt would ease visual comparison with past publications in the field, if the authors would use the conventional presentation of\nthe structure of the bacterial collagenases starting with AD on the left followed by the PD on the right etc., i.e. a horizontal\npresentation of the structure instead of the chosen “vertical one”.\nThe horizontal presentation of the collagenase has the added benefit that it is conforms better with the standard orientation\nfor (metallo-)peptidases (DOI: 10.1016/j.bbapap.2011.04.014).\nJust a suggestion, but the established notation for indicating the sequence of collagen-like peptides is easier on the eye, i.e.\n(POG)10 not POG10.\nLine 45: Please insert for better clarity “triple-helical” to yield “The triple-helical conformation is …”\nLine 51: Please insert “which” as follows in “infections and [which] have been adapted”\nLine 54: Please remove “and non-fibrillar”. Eckhard et al (2011) & Fields (2013) do not claim that bacterial collagenases can\ndigest non-fibrillar collagen; the authors only refer to fibrillar collagens I, II and III.\nLine 54: While Eckhard et al (2011) and Wang et al (2022) have provided first evidence regarding the essential role of the\nAD for collagen recognition and processing, Serwanja et al. (2024) demonstrated that the AD is responsible for collagen\nunwinding. The authors also identified residues required for collagen unwinding. Therefore, the phrase “PD-mediated\ncleavage may depend on prior AD-mediated unwinding” seems misleading regarding the processing of folded collagen.\nPlease adapt.\nLine 62: The authors should give credit to previous studies that established that bacterial collagenases and MMPs employ\nfundamentally different unwinding mechanisms.\nLine 69: Alhayet et al (2022) did not provide structural information on the conformation of M9 collagenases. Therefore,\nplease remove.\nLine 71: Please replace “unlay” with “unwind” or “untwist”.\nLine 80: The collagenase module (CM) is composed of the activator domain (AD) and the peptidase domain (PD) (Eckhard\net al (2011)). According to Eckhard et al (2011), the part that in this manuscript is referred to as “helper domain” constitutes a\nPD subdomain. If the authors can provide evidence that the helper subdomain of ColH is able to fold independently of the\nresidual PD, then it reasonable to label it as an independent domain. But otherwise please adhere to the established\nnomenclature and denote it as “helper subdomain”. Please correct the terminology in the manuscript/supplement/figures\n(either by differentiating within the PD between the “catalytic subdomain” and the “helper subdomain”, where appropriate in\nthe text/figure; or by just referring to the “PD” (not PD-HD).\nLine 85: The authors’ phrasing “The composite EM map of the AD and other domains” is suboptimal, as the term “other\ndomains” at this point implies PD, PKD1, PKD2 and CBD. Therefore, change to “The composite EM map of the CM” instead.\nIn particular, this misunderstanding can arise because the authors did not reveal any information regarding the map of the\ndomains C-terminal of the CM up to this point. A suggestion would be to move the contents of this paragraph (line 100-105)\nafter line 84, before continuing with the text in line 85.\nCan the authors comment/speculate why the CBD domain was not visible in the EM map?\nLine 85: For better local clarity please modify to “At the tip of the CM, three helices (α17 from the AD, and α18 and α21 from\nthe PD) formed …”\nFig. 1d, Inserts: Please color the helix labels differently to make them easier to find.\nFig. 1f: The shift between the frames 0 and 9 is very difficult to discriminate, in particular on the left side of the panel.\nLine 89 - line 92:\nThe authors’ conclusion regarding AD–PD interdomain flexibility is difficult to evaluate without comparing the RMSD values\nfrom the individual domain alignments to the RMSD from the overall (CM) alignment. Please clarify this point for better\ntransparency and understandability.\nLine 95: Please correct to “To assess CM flexibility,”\nLine 99: How do these low-amplitude motions compare to the “open” conformations reported by Eckhard et al./Wang et al. &\nIkeuchi et al.? Please elaborate.\nLine 128-139:\nWhile the authors emphasize the contribution of hydrophobic interactions to substrate recognition, the role of hydrogen-\nbonding interactions—also clearly present—is not discussed. The hydrophobic contacts may be of greater significance. But\ntheir significance regarding the function of the AD is not corroborated by mutational studies. The proposed binding mode\nremains incomplete without consideration of these additional hydrogen bonds.\nSpecifically, hydrogen-bonding interactions stabilize the main chain of the leading strand: Arg83:NH2 forms hydrogen bonds\nwith Gly27 and Hyp26 (2.7 Å and 2.9 Å, respectively). In addition, the position of OD1 of Hyp29 is stabilized through\nhydrogen bonds with Glu80 (2.8 Å) and Arg83 (3.0 Å). Both Glu80 and Arg83 appear in Figure 2e, but are not mentioned in\nthe text. These residues are ideally positioned to stabilize the Hyp residue at the Y-position of the collagen-like sequence,\nwhereas in ExoMiddle, the corresponding OD1 stabilization is mediated by the main-chain oxygen of Asp224.\nIn ExoMiddle, the CH–π interaction involving His236—observed in the (PPG)₁₀ and (POG)₁₀ model — is not described.\nAlthough the hydrogen-bonding network in this second exosite is less extensive than in ExoLead, interactions by Asp231,\nArg232, and Asp224 are still present. Similarly to Glu80 and Arg83 in ExoLead, Asp231 can stabilize the hydroxyl group of\nHyp26 in the Y-position. This stabilization is absent in 9LQJ, but evident in 9LYI.\nFinally, it would strengthen the manuscript to mention the findings of Serwanja et al., which identified the corresponding\nresidues to Phe37, Glu80, Arg83, Tyr87, Phe90, and Tyr184 in ExoLead in ColH as key substrate-binding residues in ColG.\nLine 135: ”Most Hyp residues remained solvent-exposed”: While this is correct, it is, nonetheless, interesting that in ExoLead\nand ExoMIddle specific pockets have evolved to stabilize the hydroxyl group of Hyp residues in the Y-position. The involved\nresidues seem to be highly conserved in the M9 family.\nLine 142: “the active-site cleft, which contracted to hold the strand tightly”. Please substantiate this observation (with a figure\nor quantify the extent of the contraction), which is – as a remark - in line with observations by Eckhard et al. (2011) NSMB\nand Eckhard et al. (2013) JBC.\nLine 144: “with conserved Gly385 and Gyl386, known as the double-glycine S1’ recognition site (Ref. 3, 17)”\nFig. 1: In Fig. 2-4, the authors provide an image illustrating the quality of the EM map by showing the EM map surrounding\nthe collagen-like peptides. Also in Fig. 1, it would be of benefit to illustrate the quality of the high-resolution EM map. In case\nthe visualization allows it, the residue sticks in the inserts, for example, could be overlaid with the EM map.\nFig. 2 g: The labeling of “Pz-peptidase” and “collagenase” could potentially confuse the reader. Different variants of ColH\nwere tested against two different substrates, i.e. Pz-peptide and collagen. Therefore, I recommend using the substrate\nnames in the legend on top of the bar graph. Please amend the figure caption stating that E416Q represents a negative\ncontrol.\nLine 147-148: The collagenase and peptidase activity of ColH were reduced by the Trp431Ala mutation. The reduction in\nturnover of both substrates is understandable given the important role of Trp431 in correctly positioning/orienting the\nsubstrate “bight” to enable binding of the residual C-terminal residues into the active-site cleft.\nLine 148: “Because Trp431 lies on a calcium-binding loop, these results further support the essential role of Ca2+\ncoordination in ColH catalysis.” This statement might not be appreciated by the audience, when they are not familiar with the\nsignificance of this calcium-binding site in bacterial collagenases. In Eckhard et al. (2013) (Ref. 17) it was proposed that\n“[d]ue to its location between the two catalytic helices, calcium binding braces the upper and lower half-domains of the\npeptidase and stabilizes thereby the zinc coordination” in the active site. Thus, calcium binding, on the one hand, stabilizes\nthe zinc in the active site, and, at the same time, the calcium-binding loop also ensures the correct positioning of Trp431 for\npre-orienting the substrate and “threading” it into the active-site cleft.\nLine 150-153:\nQuantitative comparisons would help to clarify the magnitude of the observed effects in the alanine scanning. From the\nresults shown in Fig. 2g, it can be concluded that Trp431 plays a vital role in substrate turnover for unfolded and triple-helical\nsubstrates. In case of Tyr235Ala, the mutation had a noticeable effect (~30% decrease in collagenolytic activity), whereas\nwhen all three aromatic residues of ExoMiddle were mutated an overall decrease by ~65% was observed. In comparison,\nthe Phe90Ala mutation in ExoLead alone reduced the activity by approx. 60%. Alanine mutation of the three residual\naromatic residues of this exosite led to a further decrease by 30%. This seems to suggest that the presented “three pivots”\ndiffer in their functional significance. Please elaborate more on this point. While the significance of Trp431 and Phe90\nbecome apparent from the presented data, it is difficult to ascertain whether Tyr235 is the most major determinant in\nExoMiddle, when the single alanine mutations of the other residues of this exosite are not presented. I would recommend to\nadd more mutational data regarding ExoMiddle.\nLine 154: I would recommend to rephrase the second part of the sentence for better clarity [regarding “or favours partially\nunfolded ….. POG10”].\nLine 164: “they may [have] facilitate[d] additional ColH recruitment”\nLine 178:\nThe authors state that “ExoLead-driven unwinding of POG10 was restricted because ColHMT-C bound at the C-terminus\nblocks further unravelling by ColHMT-N.” The observation that ColHMT-N was unable to unwind the bound triple-helical\ncollagen is interesting. The authors conclude that ColHMT-N could not unwind the triple helix due to the presence of\nColHMT-C, implying that the C-terminal unwinding event precludes N-terminal unwinding. Can the authors clarify whether\nthis interpretation is consistent with the conclusions drawn for the complex with (POG)12? At first glance, the two\nconclusions/statements appear to be contradictory.\nStaying with the statement “ColHMT-C bound at the C-terminus blocks further unravelling by ColHMT-N”: The authors seem\nto suggest that C-terminal unwinding induced a strain in the triple-helix which precluded a second unwinding event in close\nproximity. Or do I misinterpret the author’s statement? If yes, please provide an alternative explanation for the blockage? If\nno, is there any indication in the structural data to support the “strain hypothesis”?\nThere could be an alternative interpretation of the observed absence of unwinding in case of ColHMT-N, namely that due to\nthe absence of the middle and trailing strand at the recognition site of the leading strand (due to the mis-stagger) the local\nstructural perturbation induced by ExoLead on the leading strand could not be further sterically propagated to the middle and\ntrailing strand (which would then have been facilitated and stabilized by binding to ExoMiddle and the active-site cleft),\nthus preventing local unwinding and thus resulting in an unproductive binding event. Would this interpretation be consistent\nwith the findings regarding the complex with (POG)12?\nLines 200-201: Concerning the observed complete unfolding of (POG)₁₂ in complex with ColHMT, the authors should clarify\nwhy this observation supports the conclusion of N-to-C directional movement along the triple helix. Additionally, please\nexplain why a C-to-N directional movement would be inconsistent with the data, as this is not immediately clear from the text.\nPlease also discuss why not a similar behavior (i.e. complete unwinding of the triple helix upstream of the C-terminally\nbound ColH) was observed regarding the complex with (POG)10.\nLine 203: “shows [a] dynamic “closed” state]” Please correct.\nLine 204/Line 206: The authors correctly note that their findings “support” (line 104) the conclusion that ColH can adopt a\nratcheted state only when bound to a partially unwound substrate. In light of this, the subsequent statement that “[t]hese\nresults demonstrate that ColH exploits …” should be moderated, as the data support the claim but do not entirely\ndemonstrate it.\nLine 207: Please replace “unlaying” with “unwinding”.\nLine 211: The manuscript mentions “previous studies”; please include the appropriate citations to clarify which studies are\nbeing referenced.\nLine 212: Please change to plural, i.e. “how bacterial collagenases recognize the collagen triple helix and exploit its\ngeometry for processive stand-specific cleavage.\n@ Discussion:\nPlease elaborate on how the presented results fit with the observed “open” conformation of bacterial collagenases reported\nby Eckhard et al (Ref. 3), Wang et al. (Ref.10), Ikeuchi et al. (Ref.15), designated in this manuscript as “wide-open”\nconformation. It would be very interesting to see a figure in which the dimensions of the different opening or closing states,\nrespectively, are compared.\nLine 218: The ColHMT-N-POG10 complex represents an “unproductive” substrate binding mode (resulting probably from the\nmis-staggered triple helix at this location). While this is unfortunate, this complex nonetheless illustrates how ColH could\nutilize the captured, but unwound substrate as “rail tracks” for its one-dimensional diffusion, reminiscent of DNA-binding\nclamps. Please change the text to reflect this conclusion by the authors.\nLine 235-236: I would recommend the use of [cleavage] instead of [release]: “This captured bight is critical for translocation\nand reloading of the substrate, enabling [cleavage] of a Gly-X-Y tripeptide”.\nLine 238: “underscoring [the] mechanistic divergence between bacterial and mammalian enzymes.” Please correct.\nLines 240-248: Please clarify that the authors present and propose here a new refined model of collagen degradation by\nbacterial collagenases.\nLine 242: The term “unloads” implies active intervention by the collagenase. Was this the intended meaning? It seems more\nlikely that the cleaved products dissociate due to reduced affinity for the active-site cleft.\nLine 246: I suggest including a reference to Watanabe-Nakayama et al. (2016), who demonstrated catalytically productive\nunidirectional movement of ColG on collagen at the fibril level from C-to-N terminus, as this would provide useful context for\nthe reader.\nWhere is Reference 26 cited?\nLine 250: “was added [to remove the GST tag]”\nLine 361: Please provide buffer/elution details regarding the ion-exchange chromatography.\nLine 368: Please provide buffer details for the reconstitution of the peptide solutions.\nLine 672: It would be improve clarity if the authors elaborated why the (hypothesized) N-terminally bound ColH molecule\ncould not be observed by cryo-EM, while the C-terminally bound ColH was.\nLine 507: The method used to assess collagenolytic activity may not be perfectly able to distinguish between gelatinolytic\nand collagenolytic activity, as the assay was performed at 37°C, a temperature at which singly-accessible collagen\nmolecules on the surface of the fibril will already start to denature (doi: 10.1073/pnas.032307099).\nRegarding the reproducibility of the data, although being only a minor issue, I would like to remark that I could numerically\nnot reproduce the RMSD deviations for bond lengths and bond angles for all reported EM structures using PHENIX. Please\ncheck.\nRegarding the reported crystal structure for ColH CM, the Rwork/Rfree ratio (0.224/0.283) indicates overfitting of the density.\nPlease revise your model.\nLine 709:”as described in Extended Data Fig. 6, and Supplementary Videos 1 and 2”, these references appear to be mis-\nplaced.\nLine 710: Please correct to “activator domain” (not accessory domain).\nLine 711: Remove full stop after ColHWT.\nLine 741: “as described in Extended Data Fig. 5 and [illustrated] in Supplementary Video 3”\nLine820: Please correct to “PPG10”.\nExtended Data Fig. 1 & Extended Data Fig.4: Please provide also the intersection with FSC = 0.143 (map-to-map) and the\ncorresponding resolution."}, "author_response": "Point-by-point responses\nReviewer #1 (Remarks to the Author):\nIn this manuscript Oki H et al. describe cryo-EM and X-ray structures of the H. histolytica collagenase ColH,\na processive carboxytripeptidase. The structures presented reveal distinct states of collagen triple helix\nengagement and unwinding, providing unprecedented insight into the mechanism of collagen engagement and\ncleavage. Overall, this is a very well performed study that sheds new light on an interesting bacterial\ncollagenase with potential for future engineering or inhibitor development. For the most part the manuscript is\nvery clearly written, and should be easily understood even by a non-expert in the field. There are extensive\nsupplemental and extended data figures that are well prepared to deliver the overall story. Overall, I am highly\nsupportive of publication of this manuscript, as I feel the studies have been rigorously performed with\nappropriate analysis. I have only minor comments for the authors to consider before final publication.\nThank you for the positive evaluation of our manuscript. We greatly appreciate your generous assessment of the\nquality, clarity, and significance of our work, as well as the recognition of our careful data analysis and collection.\nWe carefully addressed the comments below and made the corresponding changes in the revised manuscript to\nfurther improve the clarity of our work.\n1. Line 98 - \"Although low in amplitude, these motions generate an entrance of ~15Å, sufficient to capture a\ntropocollagen molecule in the central cavity\". It may be nice to show a (supplemental/extended) figure with a\ntropocollagen molecule at scale next to the opening. It is hard to tell from the 3DVA alone just how large the\nopening would need to be in order for the \"clamp\" to open sufficiently to accommodate entry of tropocollagen.\nResponse: As suggested, we added Supplementary Fig. 6 showing a tropocollagen molecule [we used (POG)\n10\nas a model], viewed next to the ‘open’ conformation of the ColHWT structure (frame 9 of 3DVA component 1).\nComparison with the ‘closed’ conformation (frame 0 of 3DVA component 1) in the figure demonstrates that the\npartial opening is sufficient to allow entry of the collagen triple helix into the ColH CM ring.\nAlthough cryo-EM 3DVA reveals a cleft opening that only marginally accommodates substrate, this motion\nlikely represents the most populated trajectory under the imaged conditions. 3DVA captures dominant ensemble\nvariability and does not preclude rarer, transient excursions that exceed the observed amplitude. Such low-\noccupancy openings may be functionally critical for substrate entry, distortion, or product release but remain\nunder-represented in the cryo-EM ensemble.\nWe therefore interpret the 3DVA-derived opening as a lower bound on accessible cleft expansion rather than a\nstrict physical limit.\nSupplementary Fig. 6. Partial opening of ColHWT permits triple helix encirclement. a Enlarged view of gate\nregion in the ‘open’ ColHWT structure (frame 9 of 3DVA component 1). b Enlarged view of gate region in the\n‘closed’ ColHWT structure (frame 0 of 3DVA component 1). In both cases, a triple-helical collagen model peptide,\n(POG)₁₀, is positioned next to the gate region to represent tropocollagen.\n2. While the resolutions reported in most of the cryo-EM experiments are indeed possibly suitable for placement\nof water molecules, I am skeptical of the placement and total number of water molecules included in the refined\nstructures. In particular, in Figure 1B and 1D it is clear that the resolution in the Activator Domain is\nsignificantly lower due to inherent flexibility, yet water molecules are still modelled around the periphery of the\nactivator domain. I am skeptical that these are truly water molecules, rather than simply \"dust\" generated by\nthe automatic masking process in cryosparc. While well ordered water molecules in the protein interior with\ngood stable hydrogen bonding are likely observed in the best resolved regions, the waters that are placed at the\nperiphery of the protein (and especially in the Activator Domain) are skeptical at best. It may be worthwhile for\nthe authors to carefully review the placement of water molecules.\nResponse: Thank you for this constructive comment. As noted, the resolution of the AD region in the consensus\nmap is lower than that of other regions. This is due to the rigid-body motions of the domains mediated by the\nhinge region. Local refinement of the AD region substantially improved the EM map quality, yielding\nresolutions of 2.0–2.5 Å for most of the region, sufficient to visualise water molecules, with only terminal\nregions less clearly resolved.\nInitially, we identified water molecules solely using the sharpened cryo-EM map. To avoid potential\noversampling, we carefully re-evaluated water placement using both sharpened and unsharpened maps,\nconsidering hydrogen-bonding interactions, and focusing on regions with local resolutions better than 2.5 Å.\nBased on this reassessment, the coordinates (PDB codes: 9LRK, 9LQJ, 9WDC, 9LYI) were revised and updated\nin the PDB. Corresponding texts, tables, and figures were updated accordingly. Note that these revisions to\nwater molecule placement do not affect the conclusions of the manuscript.\n3. In addition to the point above, can the authors clearly see water molecules near the zinc active site? Including\na supplemental figure demonstrating well resolved water molecules around the zinc active site would be ideal.\nFrom the placement of these water molecules is it possible to delineate the precise positioning of the water that\nwill attack the peptide carbbonyl? This information could be very useful to those studying zinc metalloproteases\nmore generally.\nResponse: We are grateful for the opportunity to clarify the structural features of the catalytic centre including\nthe two coordinated water molecules. Accordingly, we added the relevant explanatory text (Lines 115–118 and\n223–229) to the manuscript and Supplementary Fig. 7.\nSupplementary Fig. 7. Comparison of catalytic centre of ColHWT CM in the crystal and cryo-EM structures. a\nAmino acid residues (His415, His419, and Glu447) and water molecules (w1 and w2) coordinating with the\nzinc ion in the catalytic centre of ColHWT CM in the crystal structure. 2Fo-Fc electron density maps (contoured\nat 1.0 σ) coloured light blue. b Amino acid residues (His415, His419, and Glu447) and water molecules (w1\nand w2) coordinating with the zinc ion in the catalytic centre of ColHWT CM in the cryo-EM structure. The\ncryo-EM map is contoured at 5.0 σ, and coloured light blue. The catalytic base Glu416 is also illustrated. All\ndistances are shown in Å.\nOur structural data from cryo-EM (9LRK) and crystallography (9CME), obtained at sufficient resolution to\nidentify water molecules in the active site, consistently showed two water molecules chelated to the catalytic\nZn²⁺ ion. The ion was coordinated by the conserved residues His415, His419, and the bidentate Glu447, as well\nas the two water molecules. Comparison with substrate-bound structures in which the trailing strand coordinates\nthe zinc (9LQJ, 9LRM, 9WDC, 9LYI) suggested that one of these two Zn²⁺-bound water molecules in the\nColHWT structures is displaced by the peptide carbonyl oxygen upon substrate engagement. The second water\nmolecule likely serves as the catalytic water that, once deprotonated by Glu416, attacks the carbonyl carbon of\nthe peptide bond while the zinc ion maintains a hexacoordinate geometry throughout the reaction; however, this\ncould not be observed in the present study, likely due to mutation of Glu416 to Gln.\nThe following paragraph was added between Lines 115–118.\n“At the catalytic centre, a Zn²⁺ ion was coordinated by the conserved residues, His415, His419, and the bidentate\nGlu447, as well as two water molecules (w1 and w2), as observed consistently in both the cryo-EM and X-ray\ncrystal structures, adopting hexacoordinate geometry (Supplementary Fig. 7). Neither water molecule was\npositioned to be deprotonated by Glu416.”\nThe following paragraph was added between Lines 224–230.\n“At the catalytic centre, the scissile peptide carbonyl oxygen coordinated the Zn²⁺ ion, and no water molecules\nwere observed near either the scissile bond or residue Gln416. The oxygen was located too close (3.2 Å) to the\namide oxygen of Gln416, resulting in a non-productive pentacoordinate geometry and total loss of hydrolytic\nactivities (Fig. 2f). Since the corresponding distance in a wild-type structure of Grimontia collagenase Ghcol in\ncomplex with (GPO) is 4.6 Å22, enough to evoke a substrate water molecule to the space, Glu416 in ColHWT\n2\nmay deprotonate a water molecule to initiate a nucleophilic attack.”\n4. Something seems weird in the methods section about box and pixel sizes. Line 386 - \"with a box size of 256\npixels (1.86 Å/pix) ... particles were re-extracted with a box size of 256 pixels at 0.87 Å/pix\". I think what the\nauthors mean to say is that particles were originally extracted at a box size of ~550 pixels and downsampled to\na box size of 256 pixels (1.87Å) before initial rounds of 2D classification? I don't see how the particles could\nbe extracted with the same box size and different pixel sizes unless the micrographs themselves were binned at\nsome point, or during extraction the particles were also downsampled. Please clarify in all sections throughout\nthe methods.\nResponse: We appreciate your concern regarding the clarity of the particle-extraction workflow. In the ColHWT\ndataset, particles were initially extracted after Fourier cropping (down-sampling) to 128 pixels at 1.86 Å/pixel.\nThese down-sampled particles were used for initial 2D classification, ab initio reconstruction, and\nheterogeneous refinement to remove junk particles and retain only high-quality candidates. The selected\nparticles were then re-extracted at the original box size (256 pixels) and pixel size (0.87 Å/pixel); and used for\nsubsequent homogeneous refinement, non-uniform refinement, and other subsequent analyses.\nWe have clarified in the Methods section that this same general approach—down-sampling for the initial\nextraction and classification, followed by re-extraction at the original box and pixel sizes—was employed for\nother datasets as well. Accordingly, we revised the corresponding figures (Supplementary Figs. 1, 8, 9, 14, and\n20).\n5. Line 118 - \"unwinding its terminal three triplets in an N-to-C direction\". This statement confused me when I\nfirst read it. Couldn't one argue that the actual unwinding occurs from a C-to-N direction? Figure 5 makes this\na little more clear, but at this point in the manuscript I was slightly confused by this wording and the diagram\nshown in Figure 2. Please see if revision makes sense, or this statement is accurate. As a reader I may be getting\nconfused between \"unwinding\" and \"translocation\".\nResponse: Thank you for this insightful comment and acknowledge the potential confusion caused between\n‘unwinding’ and ‘translocation’ in the original wording. The directionality of the unwinding process itself\ncannot be conclusively determined at this stage. Thus, we should have stated that ColHMT may translocate from\nthe N- to the C-terminus and unwind the three C-terminal triplets of (POG) .\n10\nposition, we did not observe it engaging in the opposite orientation or stabilising an unwound N-terminal region.\nThese results suggest that once ColHMT recognises a region that is more readily unwound [in the present case,\nthe C-terminus of (POG) ], it adopts a stable conformation at that site, becomes arrested, and does not move in\n10\nthe reverse direction thereafter.\nTo address these points, we have revised the text to distinguish between substrate translocation and unwinding.\nWe believe these changes resolve the ambiguity.\nRevised (Lines 132–139):\n“Recognition was unidirectional, with N-to-C binding of the unwound region from the front to the back face of\nColHMT (Fig. 2b, c). Neither reverse-direction recognition nor recognition involving unwinding of the central\nregion or N-terminal side of (POG) was detected in the cryo-EM analysis. Assuming that ColHMT can\n10\ntopologically entrap any site of (POG) within its CM ring, this was not initially predicted. A previous study\n10\nindicated that unwinding by M9B collagenase tends to be a local process8; therefore, ColHMT likely recognises\na readily or sufficiently unwound region during N-to-C translocation, adopts a stable conformation through\nunidirectional binding at that site, and subsequently becomes arrested.”\n6. Line 149 - it may be good to include a reference for the role of Calcium coordination in catalysis.\nResponse: Thank you for this suggestion. We have added a relevant reference (ref. 16; Eckhard et al. J. Biol.\nChem., 2013) describing the role of calcium coordination in catalysis (Line 200).\nReviewer #2 (Remarks to the Author):\nThe collagenolytic mechanism of M9 collagenases has been extensively studied. The collagen triple helix is the\nminimal structural unit of collagen, and it is generally believed that this triple helical molecule must be locally\nunwound to release a single peptide chain into the catalytic cavity before hydrolysis (2012 NSMB; 2022 NC;\nhas been lacking. In this context, the work by Oki et al. provides valuable structural and biochemical insights.\nThe authors cleverly utilized a collagen synthetic peptide that forms a stable triple helix in solution and resolved\ncryo-EM structures of M9B collagenase ColH in complex with POG10 in both its triple helical and unwound\nforms. Combined with biochemical assays, this work explores the unwinding mechanism of the collagen triple\nhelix by ColH. Despite these important contributions, there are several concerns that need to be addressed\nbefore publication.\nThank you for your thoughtful summary of our work and for recognising the significance of our structural and\nbiochemical analyses of M9 collagenase ColH. While previous studies have reported complexes primarily with\nsmall-molecule inhibitors/peptides, to our knowledge, this is the first report of a complex with a triple-helical\ncollagen substrate. We appreciate your positive evaluation of our experimental strategy, particularly the use of\nstable triple-helical collagen peptides; and the acknowledgment of the new mechanistic insights our study\nprovides into how ColH unwinds and processes the collagen triple helix.\nWe carefully addressed the concerns raised below and have made the corresponding changes in the revised\nmanuscript to further clarify and strengthen our study.\n1. From a three-dimensional structural perspective, the W431 residue is far from the catalytic center of ColH.\nHowever, as shown in Fig. 2g, the W431 mutation to alanine not only significantly reduced the activity of\ncollagen but also almost completely lost the activity of Pz peptide. There is no doubt that this phenomenon is\ncounterintuitive. Although the author provided relevant explanations in L148-L149, the significance of the W431\nresidue of ColH in triple helix unwinding or catalysis deserves to be reconsidered. The solution to this question\nwould benefit from circular dichroism spectroscopy analysis of mutant W431A and even other mutants to\ndetermine that the decreased collagen-binding ability of the mutants is caused by amino acid replacement rather\nthan structural alteration.\nIn addition, given the significance of the W431 residue in the triple helix unwinding process, the authors are\nencouraged to resolve the complex structure of W431A and POG10.\nResponse: As suggested, we performed circular dichroism spectroscopy of all mutants, including W431A\n(Supplementary Fig. 17). The obtained spectra were consistent with that of the wild-type ColH, indicating that\nthe mutations introduced in this study do not cause any detectable structural alterations.\nSupplementary Fig. 17. Circular dichroism (CD) spectra of ColHWT and its variants. Far-UV CD spectra of the\nwild-type protein and the indicated variants were measured using a Jasco J-720 spectropolarimeter at 200–250\nnm, and ellipticity is shown in millidegrees (mdeg).\nTrp431 is located close to the catalytic centre (within 10 Å from zinc to the indole ring) via the calcium-binding\nloop. Our cryo-EM analysis of the ColHMT–(POG) complex clearly demonstrated that the indole ring of\nstructural model of ColH with Pz-peptide based on the ColHMT–(POG) complex, which indicated that this\n10\ninteraction also contributes to the loss of Pz-peptide activity, as the Pro1 residue of the Pz-peptide is similarly\npositioned in the S2 subsite and interacts with Trp431 (Supplementary Fig. 18). These results from structural\nand mutational analyses further substantiate the critical role of Trp431 in ensuring stable substrate binding and\ncorrect orientation within the active-site cleft for efficient hydrolysis.\nTo explain these results, we included additional text and figures (Supplementary Figs. 17 and 18).\nRevised (Lines 211–223):\n“Strikingly, the substitution of Trp431 with alanine resulted in a dramatic decrease (~95%) in collagenolytic\nactivity and almost complete loss of enzymatic activity toward Pz-peptide, comparable to that observed for the\ncatalytically inactive E416Q mutant (Fig. 2f). Circular dichroism (CD) measurements confirmed comparable\nstructures between the mutants and ColHWT (Supplementary Fig. 17). Trp431 is located close to the catalytic\ncentre (within 10 Å from zinc to the indole ring) via the calcium-binding loop. Cryo-EM analysis of the ColHMT–\n(POG) complex clearly demonstrated that the indole ring of Trp431 plays a crucial role in stabilising Pro25\n10\n(P2) via a CH–π interaction (Fig. 2g). A structural model of ColH with Pz-peptide based on the ColHMT–(POG)\n10\ncomplex indicated that this interaction is also responsible for the loss of Pz-peptidase activity, as the Pro1 residue\nof Pz-peptide is predicted to be in the S2 subsite and interacts with Trp431 (Supplementary Fig. 18). These\nresults further substantiate the critical role of Trp431 in ensuring stable substrate binding and correct orientation\nwithin the active-site cleft for efficient hydrolysis.”\nSupplementary Fig. 18. Model structure of ColHWT–Pz-peptide complex. a Overall structure of ColHWT and\nPz-peptide complex. b Enlarged view of active site in the complex with Pz-peptide. Pz-peptide bound to PD\ncoordinates the zinc ion via the carbonyl oxygen of Leu2 (P1), and the adjacent Pro1 (P2) forms the CH–π and\nhydrophobic interactions with Trp431. The orange columns represent the carbon atoms of Pz-peptide.\nWe performed cryo-EM analysis of the complexes between ColH containing the W431A substitution and\n(POG) under the same experimental condition as the ColHMT–(POG) complex. The 2D class images (Fig.\n10 10\nR1) and reconstruction at 3.0 Å resolution yielded a structure reminiscent of ligand-free ColHWT (left panel in\nFig. R2). In contrast to ColHMT-(POG) complexes, in which a clear EM density of the trailing strand is\n10\nobserved at the active-site cleft (right panel in Fig. R2), no residual density corresponding to the peptide\nsubstrate was detected in the active-site cleft of the W431A mutant (left panel in Fig. R2). Similarly, no such\nresidual density map was observed at any of the exosites of the AD. This is likely due to the W431A mutation,\nwhich appears to have markedly reduced binding affinity.\nWhile further optimisation of conditions might provide additional insights into complex structure, this is beyond\nthe scope of the present study.\nFigure R1. 2D class images of ColH_W431A-(POG) complex.\n(POG)₁₀ complex (right panel). In the ColHMT–(POG)₁₀ complex, the EM density corresponding to the trailing\nstrand of (POG) is coloured magenta.\n10\n2. L146: What kind of interaction exists between W431 and P25 and P22? It is recommended a close-up view\nof the relative positions or interactions between relevant amino acids in Fig. 2e.\nResponse: As suggested, we added a description of the interactions to the text (Lines 192–195), along with a\nfigure (Fig. 2g) providing a close-up view of W431 and Pro25 (P2) and Pro22 (P5).\nRevised (Lines 192–195):\n“On the opposite side, Trp431 interacted with Pro25 (P2) and the Cα–H of Gly24 (P3) through CH–π\ninteractions and, together with Tyr491, interacts with Pro22 (P5) via nonpolar contacts and shape\ncomplementarity, thereby stabilising the three-residue bight (Fig. 2g).”\nFig. 2. ColH binds to unwound C-terminus of (POG) . g A magnified view of the active-site cleft around\n3. In the complex structure of ColH and POG12, why is the triple helix form of POG12 not visible? Can the\nauthor make a reasonable speculation and elaborate on it in the text? Is it related to its own properties of\nPOG12 or did the enzyme molecule ColH mediate the complete unwinding of the triple helix molecule?\nResponse: Thank you for this important question. The triple-helical form of (POG) was unexpectedly not\n12\nobserved in the ColHMT–(POG) complex. Since the triple helix of (POG) is more stable than that of (POG) ,\n12 12 10\nthis phenomenon is unlikely to result from the intrinsic properties of (POG) . Instead, as shown in Fig. 4, we\n12\npropose that two ColHMT molecules (i.e. ColHMT-N and ColHMT-C) cooperatively unwound the helix completely.\nFor (POG) , the peptide was too short for the second ColHMT molecule (ColHMT-N) to bind stably\n10\n(Supplementary Fig. 19). In contrast, for (POG) , stable binding of the second ColHMT molecule allowed\n12\nunwinding to proceed in the N-to-C direction (although C-to-N translocation may also be possible), thereby\npromoting overall unwinding.\nOnce fully unwound, the two molecules are difficult to analyse collectively owing to their orientational\nflexibility or the possible dissociation of one molecule from the unwounded peptides. Since ColHMT-N and\nColHMT-C are expected to recognise the unwounded peptide segments similarly, only an averaged structure is\nobtainable. We have revised the text (Lines 283–285) accordingly to incorporate this point.\nRevised (Lines 283–285):\n“Once fully unwound, the two molecules are difficult to analyse collectively owing to their orientational\nflexibility or possible dissociation of one molecule from the unwound peptides.”\n4. The conservation of key sites (W431, F90, Y235, etc.) related to triple helix unwinding throughout the M9\nfamily collagenases (M9A and M9B) needs to be explored.\nResponse: We appreciate the suggestion. We examined the conservation of key residues at the ExoLead and\nExoMiddle sites across the M9 family collagenases (both M9A and M9B subfamilies). Interestingly, sequence\nalignment and structural comparison revealed that the residues at ExoLead are highly conserved among M9 family\nmembers, suggesting a common mechanistic role in collagen triple-helix recognition and unwinding at this site.\nIn contrast, the residues at ExoMiddle are not as well conserved, even within the M9B subfamily.\nWe added a description of these results in the revised manuscript (Lines 147–164 in the main text and Lines\n585–591 in the methods section), and also added figures (Supplementary Figs. 12 and 13).\nRevised (Lines 147–164):\n“Partial unfolding at the C-terminus allows the leading and middle strands of (POG) to bind to two AD exosites\n10\n(ExoLead and ExoMiddle; Fig. 2b, e). Both sites engage two contiguous triplets (Pro25–…–Gly30) via hydrophobic\nclefts. Proline residues at X positions (Pro25 and Pro28) form contacts with aromatic residues—Phe37, Tyr87,\nPhe90, and Tyr184 at ExoLead; Phe176, Trp228, and Tyr235 at ExoMiddle—through hydrophobic and CH–π\ninteractions (Fig. 2e). Alanine substitution of these residues significantly reduced type I collagen hydrolysis but\nnot 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-peptide) hydrolysis, highlighting their role in\ntriple-helix recognition and unwinding (Fig. 2f). Mutations in the ExoLead residues caused a substantially greater\nreduction in collagenolytic activity than those in ExoMiddle (~90% versus ~60%), suggesting that ExoLead plays a\nmore critical role in collagen recognition and unwinding. Sequence and structural comparisons with M9A and\nM9B collagenases showed that ExoLead residues are strictly or highly conserved across these enzymes\n(Supplementary Figs. 12 and 13). Consistently, their importance for collagenolytic activity in both subfamilies\nhas been confirmed in previous mutational studies7, 8. In contrast, the aromatic residues comprising ExoMiddle in\nColH are intriguingly not as well conserved within the M9 family. In the M9B subfamily, although Y235 and\nW228 are conserved, F176 is not, whereas in M9A, only the residue equivalent to W228 is conserved. This\nrelatively low level of conservation may underlie the variability in substrate binding at this exosite.”\nRevised (Lines 585–591, Methods section):\n“Amino-acid sequences from three previously determined structures and one newly determined structure —two\nM9A collagenases (Ghcol15 and VhaC7) and two M9B collagenases (ColG5 and ColH)—were aligned using\nClustal Omega42. Structural alignment of their AD and catalytic subdomain was performed in Coot 0.9.8.93\nEL43 to identify structurally equivalent residues and refine the sequence alignment. Secondary-structure\nelements for ColH were assigned using DSSP44.”\nSupplementary Fig. 12. Amino acid sequence alignment among mature bacterial collagenases. Sequences\nof AD and PD (or catalytic subdomain) from Grimontia hollisae (M9A, Ghcol, GenBank BAK39964), Vibrio\nharveyi (M9A, VhaC, GenBank WP_047516938), and Hathewaya histolytica (M9B, ColG, GenBank\nBAA77453; and M9B, ColH, GenBank BAA06251) were aligned, and the gaps ('-') were manually adjusted by\nstructural comparison. N-terminal residues in lower case were not observed. Substrate-binding residues for the\nleading, middle, and trailing strand are coloured yellow, green, and magenta, respectively. Residues at the\ncatalytic centre are coloured red. Secondary structures of ColH are illustrated by cylinders and arrows for helices\nand sheets, respectively. The colouring is the same as the structural illustration in Fig. 1d.\nSupplementary Fig. 13. Structural comparison of the AD exosites of M9 family collagenases. a Magnified\nview of ExoLead and ExoMiddle of ColHMT–(POG) complex from H. histolytica (this study). b Magnified view\n10\nof ExoLead and ExoMiddle of ColG from H. histolytica (PDB ID: 2Y3U). c Magnified view of ExoLead and ExoMiddle\nof VhaC from V. harveyi (PDB ID: 7ESI). d Magnified view of ExoLead and ExoMiddle of Ghcol from G. hollisae\n(PDB ID: 8JT1).\n5. The proposed mechanism is mainly inferred from structural data, lacking direct kinetic or biochemical\nevidence. The discussion should explicitly acknowledge this limitation.\nResponse: We appreciate the suggestion. We added the following research limitation in the Discussion section.\nDiscussion section (Lines 343–344):\n“Nonetheless, this model is mainly inferred from structural data, lacking direct kinetic or biochemical evidence;\nit must therefore be interpreted with caution and validated in future experimental studies.”\n6. The feasibility of ColH fully wrapping around a single collagen chain within densely packed fibrils is\nquestionable. The authors should further discuss how ColH might act on native collagen substrates, going\nbeyond the simple statement that ColH targets only partially unwound or damaged regions of collagen fibrils.\nIn addition, the potential auxiliary roles of PKD and CBD domains in substrate recognition, binding, and\nunwinding should be considered to strengthen the physiological relevance of the proposed mechanism.\nResponse: We greatly appreciate the suggestion. Full-length ColH was used for cryo-EM visualisation; however,\nthe PKD2–CBD region was not resolved in the final reconstruction. In contrast, SAXS analysis revealed that\nthis collagen-binding segment extends from the catalytic module (Ohbayashi et al. Biophys. J. 104, 1538–1545,\n2013), indicating substantial conformational flexibility in solution and supporting its role as a flexible collagen-\nbinding extension. Functionally, the PKD2–CBD region binds collagen fibrils with high affinity (Kd ≈ 0.34\nµM) (Matsushita et al. J Biol Chem. 273, 3643–3648, 1998), and enhances the degradation of native collagen\nfibrils by the ColH CM (Matsushita et al. J. Biol. Chem. 273, 3643–3648, 1998). Previous studies have shown\nthat the CBD binds unidirectionally to under-twisted regions of the triple helix (Philominathan et al. J. Biol.\nChem. 284, 10868–10876, 2009; Philominathan et al. Protein Sci. 21, 1554–1565, 2012), whereas the PKD\ndomain promotes fibril expansion and swelling without disrupting the integrity of the triple helix (Wang et al.\nJ. Biol. Chem. 285, 14285–14291, 2010).\nThese findings suggest that, within densely packed fibrils where steric constraints prevent the catalytic module\nfrom fully wrapping around a single tropocollagen molecule, the PKD2–CBD segment may function as an\nauxiliary tether that recognises locally distorted or partially unbundled regions of collagen. By transiently\nanchoring ColH to such hypersensitive sites, the PKD2–CBD region may guide the triple helix into the catalytic\ncleft, facilitating localised unwinding and cleavage. This auxiliary targeting mechanism is analogous to how\ntranscription factors position RNA polymerase on DNA and likely enhances catalytic efficiency by enabling\nsurface diffusion and selective engagement of vulnerable sites, rather than relying solely on random encounters\nwith an insoluble substrate. In addition, consistent with a previous study (Wang et al. Nat. Commun. 13, 566,\n2022), PKD2–CBD may bind to regions that promote PD-mediated telopeptide cleavage, thereby exposing the\ntropocollagen for subsequent enzymatic processing.\nWe added a description of this valuable topic in the revised manuscript (Lines 344–353 in the discussion section).\nRevised (Lines 346–354):\n“Additionally, the present study leaves the unresolved question of how ColH overcomes steric constraints within\ndensely packed collagen fibrils. The PKD2–CBD region of the collagenase may act as an auxiliary tether that\nrecognises locally distorted or partially unbundled collagen regions27, 28. By transiently anchoring ColH to these\nhypersensitive sites, the PKD2–CBD region may help position the triple helix into the catalytic cleft, thereby\nfacilitating localised unwinding and cleavage. This auxiliary targeting strategy may enhance catalytic efficiency\nby enabling surface diffusion and selective engagement of vulnerable sites, rather than relying solely on random\nencounters with an insoluble substrate. Nevertheless, the precise mechanism by which ColH achieves efficient\nfibril hydrolysis remains unclear, which will require further investigation.”\nReviewer #3 (Remarks to the Author):\nThe manuscript by Oki and colleagues entitled \"Bacterial Collagenase Harnesses Collagen Geometry for\nProcessive Cleavage” reports on a study that investigates the mechanism of how bacterial collagenases unwind\nand cleave collagen. Collagen’s triple-helical fold and high content of (hydroxy-)proline render the molecule\nextremely stable and resistant to enzymatic proteolysis within the context of the extracellular matrix. To\novercome this difficulty, true collagenases, i.e. those enzymes that process natively folded triple-helical collagen,\nmust unwind the molecule locally to make the cleavage sites accessible to the enzyme. Based on high-resolution\ncryo–electron microscopy findings the authors demonstrate that ColH encircles a single collagen triple helix\nvia its AD and PD domains. Via the hydrophobic AD exosites the triple helix is eventually stripped of its\nstabilizing hydration shell by the enzymes, and the substrate’s leading and middle strand are bound\nand sterically distorted by binding to the two AD exosites. The displaced trailing strand is threaded via “bight”\nformation mediated by Trp431 into the active-site cleft. Exploiting collagen’s geometry for continuous\ndegradation, the authors propose that small-amplitude AD-PD movements, i.e. cycling between “ratcheted”\nand “closed” stated, allow for triplet-by-triplet translocation and its stepwise processing.\nThe present work is of great importance to the field and beyond, and deserves publication in Nature\nCommunications.\nMerely, the following minor issues should be dealt with in a revised version of the manuscript before prior to\nacceptance for publication.\nThank you for the careful and detailed review of our work and for recognising the significance of our findings,\nthat is, the mechanism of collagen triple-helix unwinding and processive cleavage by ColH. We are grateful for\nthe positive evaluation of our study and its contribution to the field. We carefully addressed the issues raised\nbelow and made the corresponding changes in the revised manuscript to further clarify and strengthen our work.\nThe numbering of the references is not chronological. Please correct.\nResponse: Thank you for pointing this out. The references have been renumbered and arranged in chronological\norder in the revised manuscript.\nIt would ease visual comparison with past publications in the field, if the authors would use the conventional\npresentation of the structure of the bacterial collagenases starting with AD on the left followed by the PD on\nthe right etc., i.e. a horizontal presentation of the structure instead of the chosen “vertical one”.\nThe horizontal presentation of the collagenase has the added benefit that it is conforms better with the standard\norientation for (metallo-)peptidases (DOI: 10.1016/j.bbapap.2011.04.014).\nResponse: In preparing the draft, we carefully considered the consistency of the figure orientation with that of\nprevious publications. However, it was not possible to clearly represent the enzyme motions in the conventional\norientation. Therefore, we deliberately chose ‘vertical views’ for this manuscript.\nJust a suggestion, but the established notation for indicating the sequence of collagen-like peptides is easier\non the eye, i.e. (POG)10 not POG10.\nResponse: We changed the notation of PPG10 and POG10 to (PPG) and (POG) , respectively, in the revised\n10 10\nmanuscript.\nLine 45: Please insert for better clarity “triple-helical” to yield “The triple-helical conformation is …”\nResponse: We apologise for the lack of clarity. The manuscript has been revised to address this, and ‘triple-\nhelical’ has been inserted so that the sentence now reads “The triple-helical conformation is …” (Line 52).\nLine 51: Please insert “which” as follows in “infections and [which] have been adapted”\nResponse: The manuscript has been revised accordingly, and ‘which’ has been inserted into the corresponding\nsentence (Line 58).\nLine 54: Please remove “and non-fibrillar”. Eckhard et al (2011) & Fields (2013) do not claim that bacterial\ncollagenases can digest non-fibrillar collagen; the authors only refer to fibrillar collagens I, II and III.\nResponse: Thank you for pointing this out. We removed ‘and non-fibrillar’ from the sentence.\nLine 54: While Eckhard et al (2011) and Wang et al (2022) have provided first evidence regarding the essential\nrole of the AD for collagen recognition and processing, Serwanja et al. (2024) demonstrated that the AD is\nresponsible for collagen unwinding. The authors also identified residues required for collagen unwinding.\nTherefore, the phrase “PD-mediated cleavage may depend on prior AD-mediated unwinding” seems misleading\nregarding the processing of folded collagen. Please adapt.\nResponse: Thank you for the careful review and constructive feedback. To better reflect the current\nunderstanding in the literature, we revised the sentence as follows:\nLine 61:\n“PD-mediated cleavage depends on prior AD-mediated unwinding”\nLine 62: The authors should give credit to previous studies that established that bacterial collagenases and\nMMPs employ fundamentally different unwinding mechanisms.\nResponse: Thank you for pointing this out. We added citations (refs. 5, 7, 8, 9, 10) in the revised manuscript\n(Line 69) to acknowledge that bacterial collagenases and MMPs employ fundamentally different unwinding\nmechanisms, as evidenced in previous studies.\nLine 69: Alhayet et al (2022) did not provide structural information on the conformation of M9 collagenases.\nTherefore, please remove.\nResponse: Thank you for pointing this out. The manuscript has been revised accordingly.\nLine 71: Please replace “unlay” with “unwind” or “untwist”.\nResponse: The word ‘unlay’ was chosen because the three strands are unwound and fixed in three different\nspatial positions.\nLine 80: The collagenase module (CM) is composed of the activator domain (AD) and the peptidase domain\n(PD) (Eckhard et al (2011)). According to Eckhard et al (2011), the part that in this manuscript is referred to as\n“helper domain” constitutes a PD subdomain. If the authors can provide evidence that the helper subdomain\nof ColH is able to fold independently of the residual PD, then it reasonable to label it as an independent domain.\nBut otherwise please adhere to the established nomenclature and denote it as “helper subdomain”. Please\ncorrect the terminology in the manuscript/supplement/figures (either by differentiating within the PD between\nthe “catalytic subdomain” and the “helper subdomain”, where appropriate in the text/figure; or by just\nreferring to the “PD” (not PD-HD).\nResponse: Thank you for the detailed comment and noting the issue regarding the ‘helper domain’ nomenclature.\nWe agree that, according to Eckhard et al. (2011), this region is formally considered a subdomain of the PD.\nIn the revised manuscript, we have adopted the established terminology and now refer to this region as the\n‘helper subdomain’ of the PD throughout the text, figures, and supplementary information. Where relevant, we\nhave differentiated the ‘catalytic subdomain’ within the PD to improve clarity.\nThis important suggestion has helped us ensure consistent and accurate use of structural nomenclature, thank\nyou.\nLine 85: The authors’ phrasing “The composite EM map of the AD and other domains” is suboptimal, as the\nterm “other domains” at this point implies PD, PKD1, PKD2 and CBD. Therefore, change to “The composite\nEM map of the CM” instead.\nIn particular, this misunderstanding can arise because the authors did not reveal any information regarding the\nmap of the domains C-terminal of the CM up to this point. A suggestion would be to move the contents of this\nparagraph (line 100-105) after line 84, before continuing with the text in line 85.\nResponse: Thank you for pointing this out. We moved the contents of the paragraph and revised the sentences\nas shown below (Lines 89–101).\nRevised (Lines 89–101):\n“Focused refinement improved AD map quality, revealing a rigid structure with orientational flexibility relative\nto the other domains (Supplementary Fig. 1). Our cryo-EM analysis revealed a clear density for polycystic\nkidney disease-like domain 1 (PKD1) but not PKD2 or the collagen-binding domain (CBD), likely owing to\nlinker flexibility that averages out their densities in the EM reconstruction.\nThe composite EM map of the AD and PD-PKD1 regions revealed a ‘closed’ CM conformation (Fig. 1d\nand Supplementary Figs. 1 and 2). At the junction between AD and PD, three helices (α17, α18, and α21) formed\na hinge via hydrophobic interactions, whereas gate-region interactions on the opposite side reinforced the ring\n(Fig. 1d). PKD1 interacts with the PD in a calcium-dependent manner (Fig. 1d). A previous small-angle X-ray\nscattering experiment showed that calcium depletion produces an elongated apo ColH envelope17. Thus, the\nrigid PKD1 conformation may stabilise the overall structure and restrict PKD2-CBD orientation.”\nCan the authors comment/speculate why the CBD domain was not visible in the EM map?\nResponse: Thank you for this question. The CBD, as well as the PKD2 domain, was not resolved in the current\nEM map, likely due to their inherent flexibility relative to the CM-PKD1. Such mobility would result in weak\nor averaged-out density in the EM reconstruction. We added a statement (Lines 91–94) in the revised manuscript\nto address this point.\nRevised (Line 91–94):\n“Our cryo-EM analysis revealed a clear density for polycystic kidney disease-like domain 1 (PKD1) but not\nPKD2 or the collagen-binding domain (CBD), likely owing to linker flexibility that averages out their densities\nin the EM reconstruction.”\nLine 85: For better local clarity please modify to “At the tip of the CM, three helices (α17 from the AD, and α18\nand α21 from the PD) formed …”\nResponse: Thank you for the suggestion. We have modified the text accordingly to read “At the junction between\nAD and PD, three helices (α17 from the AD, and α18 and α21 from the PD) formed …” in the revised manuscript\n(Lines 96–98). The hinge region is located at the junction between AD and PD; therefore, we changed the term\n‘tip’ to ‘junction.’\nFig. 1d, Inserts: Please color the helix labels differently to make them easier to find.\nResponse: Thank you for this suggestion. In the revised manuscript, we have coloured the helix labels in the\ninserts of Fig. 1d in the same way as the domain structure to improve their visibility.\nFig. 1f: The shift between the frames 0 and 9 is very difficult to discriminate, in particular on the left side of the\npanel.\nResponse: We apologise for the lack of clarity. To address this issue, we prepared an enlarged view of the gate\nregion (Fig. 1f). This magnified representation allows the structural features and relative differences to be more\nclearly visualised. The corresponding figure and caption have been updated accordingly.\nLine 89 - line 92:\nThe authors’ conclusion regarding AD–PD interdomain flexibility is difficult to evaluate without comparing the\nRMSD values from the individual domain alignments to the RMSD from the overall (CM) alignment. Please\nclarify this point for better transparency and understandability.\nResponse: In the revised manuscript, we discuss the interdomain flexibility between the AD and PD based on\nthe B-factors obtained from crystallographic analysis, rather than using RMSD. As shown in Supplementary\nFig. 4, the B-factors gradually increase from the C- to N-terminus of the AD, suggesting domain movements\nmediated by the hinge region. These observations are consistent with the 3DVA of cryo-EM data.\nWe added a description of these results in the revised manuscript (Lines 101–106 in the main text, and in the\nmethods section) and also added the figures (Supplementary Fig. 4).\nRevised (Lines 101–106):\n“We also performed X-ray crystallography of ColHWT CM at room temperature, which yielded a 2.70 Å structure\nessentially identical to the cryo-EM model, with a root-mean-square deviation of 1.9 Å for Cα atoms\n(Supplementary Fig. 3 and Supplementary Table 2). Notably, crystallographic B-factors increase progressively\nfrom the C-terminus to the N-terminus of the AD, suggesting hinge-mediated flexibility between the AD and\nPD (Supplementary Fig. 4).”\nSupplementary Fig. 4. Graphical representations of ColHWT CM flexibility. a Colour mapping of B-facotrs\non the crystal structure of ColHWT CM. The data use a colour scale from 12.75 Å2 (blue) to 122.46 Å2 (red). b\nPlotting of B-factors along the residue number. In the AD, B-factors increased linearly toward the N-terminus.\nLine 95: Please correct to “To assess CM flexibility,”\nResponse: Thank you for this comment. The text has been corrected as suggested to read “To assess CM\nflexibility,” in the revised manuscript (Line 109).\nLine 99: How do these low-amplitude motions compare to the “open” conformations reported by Eckhard et\nal./Wang et al. & Ikeuchi et al.? Please elaborate.\nResponse: Thank you for this valuable comment. Our 3DVA revealed, somewhat unexpectedly, that ColH is\nstabilised in a closed conformation and does not adopt the wide-open structures observed in ColG and other M9\nfamily collagenases. Although a closed conformation of ColG has not yet been reported, these structural\ndifferences may contribute to the distinct substrate specificities of these enzymes. We added sentences (Lines\n315–320) explicitly addressing this point in the revised manuscript.\nRevised (Lines 315–320):\n“These residues may also help ColH recognise vulnerable sites in tropocollagen as it slides from the N- to C-\nterminus (Fig. 5c and d). Notably, the dynamic ‘closed’ conformation of ColH is in stark contrast to that of\nhomologous enzymes such as ColG and some members of the M9A subfamily, which adopt more widely opened\nAD–PD arrangements (Supplementary Fig. 21)5, 7, 15. The closed-biased architecture of ColH may enhance its\nability to capture rare, thermally populated local fluctuations of the collagen triple helix, thereby promoting\nactive unwinding.”\nLine 128-139:\nWhile the authors emphasize the contribution of hydrophobic interactions to substrate recognition, the role of\nhydrogen-bonding interactions—also clearly present—is not discussed. The hydrophobic contacts may be of\ngreater significance. But their significance regarding the function of the AD is not corroborated by mutational\nstudies. The proposed binding mode remains incomplete without consideration of these additional hydrogen\nbonds.\nSpecifically, hydrogen-bonding interactions stabilize the main chain of the leading strand: Arg83:NH2 forms hydrogen bonds\nwith Gly27 and Hyp26 (2.7 Å and 2.9 Å, respectively). In addition, the position of OD1 of Hyp29 is stabilized through\nhydrogen bonds with Glu80 (2.8 Å) and Arg83 (3.0 Å). Both Glu80 and Arg83 appear in Figure 2e, but are not mentioned in\nthe text. These residues are ideally positioned to stabilize the Hyp residue at the Y-position of the collagen-like sequence,\nwhereas in ExoMiddle, the corresponding OD1 stabilization is mediated by the main-chain oxygen of Asp224.\nIn ExoMiddle, the CH–π interaction involving His236—observed in the (PPG)₁₀ and (POG)₁₀ model — is not\ndescribed. Although the hydrogen-bonding network in this second exosite is less extensive than in ExoLead,\ninteractions by Asp231, Arg232, and Asp224 are still present. Similarly to Glu80 and Arg83 in ExoLead, Asp231\ncan stabilize the hydroxyl group of Hyp26 in the Y-position. This stabilization is absent in 9LQJ, but evident in\n9LYI.\nFinally, it would strengthen the manuscript to mention the findings of Serwanja et al., which identified the\ncorresponding residues to Phe37, Glu80, Arg83, Tyr87, Phe90, and Tyr184 in ExoLead in ColH as key\nsubstrate-binding residues in ColG.\nResponse: Thank you for this constructive comment. We agree that hydrogen-bonding interactions are also\npresent at the binding interface. As suggested, we added descriptions of these hydrogen bonds and their potential\nrole in substrate recognition in the revised manuscript.\nOur sequence alignment and structural comparison (Supplementary Figs. 12 and 13) revealed that, although\nmost of the residues involved in these hydrogen-bonding or CH-π interactions (such as His236 and Asn232) are\nnot conserved across the M9 collagenase family, Arg83, which stabilises the main chain of the leading strand,\nis strictly conserved in both the M9A and M9B subfamilies. In addition to the conserved aromatic residues,\nthe critical role of this arginine in collagenolytic activity has been confirmed in previous mutational studies\n(Serwanja et al. Proc. Natl. Acad. Sci. U.S.A. 121, e2321002121, 2024; Wang et al. Nat. Commun. 13, 566,\n2022). \nWe added a discussion (Lines 165–175 in the Results and Lines 308–311 in the Discussion section) of these\npoints in the revised manuscript and cited the relevant references (refs. 7 and 8).\nRevised (Lines 165–175):\n“Besides the aromatic interactions, several hydrogen-bonding interactions were observed (Fig. 2e). At ExoLead,\nsuch interactions stabilise the conformation of the leading strand: the main-chain carbonyls of Hyp26 and Gly27\nform hydrogen bonds with Arg83, while the OD1 atom of Hyp29 form hydrogen bonds with Glu80 and Arg83.\nAlthough hydrogen-bonding networks at ExoMiddle are less extensive, an interaction between the carbonyl group\nof Hyp26 and Asn232 as well as OD1 of Hyp29 and carbonyl group of Asp224 stabilises the middle strand.\nWhile these interactions further strengthen peptide binding, the residues are not strictly conserved within the\nM9 family, with the exception of Arg83 (Supplementary Figs. 12 and 13). The importance of arginine at this\nposition for substrate recognition in M9A and M9B collagenases has been demonstrated in mutational studies7,\n8, consistent with our structural observation that Arg83 engages and stabilises the main chain of the leading\nstrand.”\nRevised (Lines 308–311):\n“Recognition relies on two AD exosites, primarily ExoLead, which orient consecutive Gly–Pro–Yaa triplets via\nhydrophobic and CH–π interactions. Although less extensive, several hydrogen-bonding interactions,\nparticularly those involving the conserved Arg83 at ExoLead, contribute to peptide strand positioning.”\nLine 135: ”Most Hyp residues remained solvent-exposed”: While this is correct, it is, nonetheless, interesting\nthat in ExoLead and ExoMIddle specific pockets have evolved to stabilize the hydroxyl group of Hyp residues\nin the Y-position. The involved residues seem to be highly conserved in the M9 family.\nResponse: One of our key findings is that the AD of ColH recognises the Gly–Pro–Yaa–Gly–Pro–Yaa motif. In\nthe CoHMT–(POG) complex structure, most Hyp residues are oriented toward the solvent or face large pockets,\n10\nand some form hydrogen bonds with several residues (such as Glu80, Arg83, Asp231); however, most of these\ninteracting residues, except Arg83, are not strictly conserved (Supplementary Figs. 12 and 13). Therefore, these\npockets, while capable of accommodating Hyp, are also likely to recognise other residues. These observations\nare consistent with previous proteomics data showing that clostridial collagenases exhibit selectivity for Gly–\nPro–Yaa triplets (Eckhard et al., J. Proteomics, 2013).\nIn light of these considerations, we revised the relevant sections of the manuscript (Lines 176–182).\nRevised (Lines 176–182):\n“Most Hyp residues remained solvent-exposed and contributed little to binding, consistent with the cryo-EM\ndata of ColHMT bound to [(Pro–Pro–Gly)₁₀]₃ [or (PPG) ] (Supplementary Fig. 14 and Supplementary Table 1),\n10\nwhich showed a binding mode similar to that of the ColHMT–(POG) complex (Supplementary Fig. 15). These\n10\nresults suggest that ColH has evolved to recognise Gly–Pro–Yaa–Gly–Pro–Yaa motifs in native collagen via its\nhydrophobic exosites, as evidenced in previous proteomics analyses demonstrating the substrate specificity of\nclostridial collagenases for Gly–Pro–Yaa triplets20.”\nLine 142: “the active-active-site cleft, which contracted to hold the strand tightly”. Please substantiate this\nobservation (with a figure or quantify the extent of the contraction), which is – as a remark - in line with\nobservations by Eckhard et al. (2011) NSMB and Eckhard et al. (2013) JBC.\nResponse: Thank you for this helpful comment. To substantiate the contraction of the active-site cleft, we added\na figure illustrating the extent of cleft contraction upon substrate binding (Supplementary Fig. 16). For\ncomparison, we also presented the structures of ColG and ColT, for which both inhibitor-bound and inhibitor-\nfree forms have been reported. We further note that this observation is consistent with previous reports (Eckhard\net al. Nat. Struct. Mol. Biol. 18, 1109–1114, 2011; Eckhard et al. J. Biol. Chem. 288, 20184–20194, 2013),\nand have cited these studies accordingly in the revised text (Lines 182–188).\nRevised (Lines 183–189):\n“Although the leading and middle strands followed a triple-helical axis with minor deviation, the trailing strand\nveered sharply, threading the three triplets at its C-terminus (Gly21–…–Gly30; P6 to P4′) into the active-site\ncleft (Fig. 2c, e). This distortion is critical for Zn²⁺-mediated chelation of the scissile carbonyl. As observed in\nthe inhibitor-bound structures of other M9B collagenases5, 16, the active-site cleft embraced by the N- and C-\nterminal half-domains of the PD catalytic subdomain was contracted as much as 3 Å, holding the trailing strand\ntightly (Fig. 2d and Supplementary Fig. 16).”\nSupplementary Fig. 16. Ligand-induced contraction of PD catalytic subdomain in M9B collagenases.\nSuperpositions of ligand-bound and ligand-free PD structures for ColH (top), ColG (bottom left), and ColT from\nClostirdium tetani (bottom right). In each case, the superposition was performed using the N-terminal half-\ndomain, and contraction of the C-terminal half-domain relative to the well-overlapped N-terminal half-domain\nis indicated by arrows. The N-terminal and C-terminal half domains are coloured grey and cyan, respectively.\nThe helper subdomain is coloured blue.\nLine 144: “with conserved Gly385 and Gyl386, known as the double-glycine S1’ recognition site (Ref. 3, 17)”\nResponse: Thank you for the suggestion. We have modified the text to read “with conserved Gly385 and Gly386\non the β4 edge strand, known as the double-glycine S1’ recognition site5, 16” in the revised manuscript\n(Lines 189–190).\nFig. 1: In Fig. 2-4, the authors provide an image illustrating the quality of the EM map by showing the EM map\nsurrounding the collagen-like peptides. Also in Fig. 1, it would be of benefit to illustrate the quality of the high-\nresolution EM map. In case the visualization allows it, the residue sticks in the inserts, for example, could be\noverlaid with the EM map.\nResponse: We appreciate the helpful comment. In this study, ColHWT shown in Fig. 1 was analysed at near-\natomic resolution (2.3 Å), allowing us to clearly visualise even the water molecules around the active site.\nTherefore, as also suggested by another reviewer, we prepared and included an EM map focused on the active\nsite (Supplementary Fig. 7). We believe that this more clearly demonstrates the quality of our analysis.\nFig. 2 g: The labeling of “Pz-peptidase” and “collagenase” could potentially confuse the reader. Different\nvariants of ColH were tested against two different substrates, i.e. Pz-peptide and collagen. Therefore, I\nrecommend using the substrate names in the legend on top of the bar graph. Please amend the figure caption\nstating that E416Q represents a negative control.\nResponse: Thank you for noting our mistakes in preparing Fig. 2g (Fig. 2f in the revised manuscript). We\nremoved the explanations of Pz-peptidase and collagenase assays from the graph and described them in the\nlegend. We used reaction buffers as the negative controls for the assays.\nLine 147-148: The collagenase and peptidase activity of ColH were reduced by the Trp431Ala mutation. The\nreduction in turnover of both substrates is understandable given the important role of Trp431 in correctly\npositioning/orienting the substrate “bight” to enable binding of the residual C-terminal residues into the active-\nsite cleft.\nResponse: We agree with the interpretation and have rephrased the relevant sentence in the revised manuscript\nto highlight the crucial role of Trp431 in correctly positioning and orienting the substrate ‘bight,’ thereby\npromoting proper binding of the residual C-terminal residues within the active-site cleft (Lines 221–223).\nRevised (Lines 221–223):\n“These results further substantiate the critical role of Trp431 in ensuring stable substrate binding and correct\norientation within the active-site cleft for efficient hydrolysis.”\nLine 148: “Because Trp431 lies on a calcium-binding loop, these results further support the essential role of\nCa2+ coordination in ColH catalysis.” This statement might not be appreciated by the audience, when they are\nnot familiar with the significance of this calcium-binding site in bacterial collagenases. In Eckhard et al. (2013)\n(Ref. 17) it was proposed that “[d]ue to its location between the two catalytic helices, calcium binding braces\nthe upper and lower half-domains of the peptidase and stabilizes thereby the zinc coordination” in the active\nsite. Thus, calcium binding, on the one hand, stabilizes the zinc in the active site, and, at the same time, the\ncalcium-binding loop also ensures the correct positioning of Trp431 for pre-orienting the substrate and\n“threading” it into the active-site cleft.\nResponse: Thank you for this constructive comment. In the revised manuscript, we note that, as proposed by\nEckhard et al. (2013, ref. 16), calcium binding braces the upper and lower half-domains of the peptidase, thereby\nstabilising zinc coordination in the active site. In addition, we highlight that the calcium-binding loop also\npositions Trp431, facilitating pre-orientation of the substrate and its proper threading into the active-site cleft\n(Lines 195–200).\nRevised (Line 195–200):\n“Notably, Trp431 lies on a calcium-binding loop located between the N- and C-terminal half-domains, where a\nconserved glutamate side-chain (Glu390 in ColH) anchors the edge strand and calcium, thereby bracing the two\nhalf-domains (Fig. 2g). Consequently, calcium binding not only stabilises zinc coordination in the active site\nbut also ensures the proper positioning of Trp431. These observations support the essential role of Ca²⁺\ncoordination in ColH catalysis16.”\nLine 150-153:\nQuantitative comparisons would help to clarify the magnitude of the observed effects in the alanine scanning.\nFrom the results shown in Fig. 2g, it can be concluded that Trp431 plays a vital role in substrate turnover for\nunfolded and triple-helical substrates. In case of Tyr235Ala, the mutation had a noticeable effect (~30% decrease in collagenolytic activity), whereas\nwhen all three aromatic residues of ExoMiddle were mutated an overall decrease by ~65% was observed. In comparison,\nthe Phe90Ala mutation in ExoLead alone reduced the activity by approx. 60%. Alanine mutation of the three residual\naromatic residues of this exosite led to a further decrease by 30%. This seems to suggest that the presented “three pivots”\ndiffer in their functional significance. Please elaborate more on this point. While the significance of Trp431 and Phe90\nbecome apparent from the presented data, it is difficult to ascertain whether Tyr235 is the most major determinant in\nExoMiddle, when the single alanine mutations of the other residues of this exosite are not presented. I would recommend to\nadd more mutational data regarding ExoMiddle.\nResponse: Thank you for this important comment. We agree that quantitative comparisons provide insight into\nthe relative functional contributions of the aromatic residues in the ExoLead and ExoMiddle sites.\nAs noted, the qualitative comparison indicated differences in the contributions of the three aromatic residues,\nwith Tyr235 in ExoMiddle having a weaker effect than Phe90 in ExoLead. This trend was also observed in the four-\nresidue ExoLead mutant (F37A/Y87A/F90A/Y184A) and three-residue ExoMiddle mutant\n(F176A/W228A/Y235A), suggesting that the overall contribution of ExoMiddle is smaller than that of ExoLead.\nThis is also consistent with our sequence and structural comparisons showing that the residues in ExoMiddle are\nnot conserved, reflecting the variation in substrate recognition at this exosite (Supplementary Figs. 12 and 13).\nGiven the relatively minor contribution of ExoMiddle residues to collagenase activity, we did not perform further\nmutational experiments; however, the manuscript has been revised to address these points (Lines 147–164 and\n200–209).\nRevised (Lines 147–164):\n“Partial unfolding at the C-terminus allows the leading and middle strands of (POG) to bind to two AD exosites\n10\n(ExoLead and ExoMiddle; Fig. 2b, e). Both sites engage two contiguous triplets (Pro25–…–Gly30) via hydrophobic\nclefts. Proline residues at X positions (Pro25 and Pro28) form contacts with aromatic residues—Phe37, Tyr87,\nPhe90, and Tyr184 at ExoLead; Phe176, Trp228, and Tyr235 at ExoMiddle—through hydrophobic and CH–π\ninteractions (Fig. 2e). Alanine substitution of these residues significantly reduced type I collagen hydrolysis but\nnot 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-peptide) hydrolysis, highlighting their role in\ntriple-helix recognition and unwinding (Fig. 2f). Mutations in the ExoLead residues caused a substantially greater\nreduction in collagenolytic activity than those in ExoMiddle (~90% versus ~60%), suggesting that ExoLead plays a\nmore critical role in collagen recognition and unwinding. Sequence and structural comparisons with M9A and\nM9B collagenases showed that ExoLead residues are strictly or highly conserved across these enzymes\n(Supplementary Figs. 12 and 13). Consistently, their importance for collagenolytic activity in both subfamilies\nhas been confirmed in previous mutational studies7, 8. In contrast, the aromatic residues comprising ExoMiddle in\nColH are intriguingly not conserved within the M9 family. In the M9B subfamily, although Y235 and W228 are\nconserved, F176 is not, whereas in M9A, only the residue equivalent to W228 is conserved. This relatively low\nlevel of conservation may underlie the variability in substrate binding at this exosite.”\nRevised (Lines 201–210):\n“When viewed from the front face of ColHMT, three aromatic residues, Phe90, Tyr235, and Trp431, engaged the\ninteracting triplets as aromatic pivots marking the transition from a helical to unwound structure (Fig. 2h). These\nresidues may enhance local bending to promote strand unwinding or favours partially unfolded triple-helical\nstructures, facilitating recognition of the frayed C-terminal region of (POG) 21. Substitution of Phe90 greatly\n10\nreduced collagenolytic activity, whereas Tyr235 mutation led to a more modest decrease (~60% versus ~20%;\nFig. 2f). Although Tyr235 is not conserved in the M9A family (Supplementary Fig. 12), a phenylalanine residue\nthat may serve a similar function is positioned nearby (Supplementary Fig. 13). This suggests that an aromatic\nresidue at this position supports the binding and stabilisation of the unwound collagen triple helix.”\nLine 154: I would recommend to rephrase the second part of the sentence for better clarity [regarding “or\nfavours partially unfolded. POG10”].\nResponse: Thank you for this note. The corresponding section has been rephrased as follows:\nRevised (Lines 203–205):\n“These residues may enhance local bending to promote strand unwinding or favours partially unfolded triple-\nhelical structures, facilitating recognition of the frayed C-terminal region of (POG) 21.”\nResponse: Thank you for the suggestion. We have made the corrections in the revised manuscript (Line 239).\nLine 178:\nThe authors state that “ExoLead-driven unwinding of POG10 was restricted because ColHMT-C bound at the\nC-terminus blocks further unravelling by ColHMT-N.” The observation that ColHMT-N was unable to unwind\nthe bound triple-helical collagen is interesting. The authors conclude that ColHMT-N could not unwind the\ntriple helix due to the presence of ColHMT-C, implying that the C-terminal unwinding event precludes N-\nterminal unwinding. Can the authors clarify whether this interpretation is consistent with the conclusions drawn\nfor the complex with (POG)12? At first glance, the two conclusions/statements appear to be contradictory.\nStaying with the statement “ColHMT-C bound at the C-terminus blocks further unravelling by ColHMT-N”:\nThe authors seem to suggest that C-terminal unwinding induced a strain in the triple-helix which precluded a\nsecond unwinding event in close proximity. Or do I misinterpret the author’s statement? If yes, please provide\nan alternative explanation for the blockage? If no, is there any indication in the structural data to support the\n“strain hypothesis”?\nThere could be an alternative interpretation of the observed absence of unwinding in case of ColHMT-N, namely\nthat due to the absence of the middle and trailing strand at the recognition site of the leading strand (due to the\nmis-stagger) the local structural perturbation induced by ExoLead on the leading strand could not be further\nsterically propagated to the middle and trailing strand (which would then have been facilitated and stabilized\nby binding to ExoMiddle and the active-site cleft), thus preventing local unwinding and thus resulting in an\nunproductive binding event. Would this interpretation be consistent with the findings regarding the complex with\n(POG)12?\nResponse: We apologise for the lack of clarity. We propose that this inhibitory effect is not due to the unwinding\nof ColHMT-C, but rather that ColHMT-C acts as a steric impediment in the 2:1 ColHMT–(POG) complex, thereby\n10\nhindering further unwinding by N-terminally bound ColH-N.\nTo clarify this point, we added Supplementary Fig. 19 illustrating the apparent steric hindrance between the\nPKD1 domain of ColHMT-N and the CM of ColHMT-C in the 2:1 ColHMT-(POG) complex.\nColHMT–(POG)₁₀ complex. In the complex, ColHMT molecules are depicted as surface representations. The\nPKD1 of ColHMT-N, which is likely involved in the steric hindrance, is indicated by an arrowhead.\nAs noted, we observed that ColHMT-N binds at the N-terminus and induces local structural changes (Fig. 2),\nparticularly in the mis-staggered leading strand via ExoLead; however, these changes cannot propagate further,\nas they are insufficient to allow the middle and trailing strands to engage stably with ExoMiddle and the active\nsite, respectively, due to the aforementioned steric hindrance. We have included a sentence (Lines 253–258)\nin the revised manuscript to clarify this situation. Note that this steric hindrance would not occur for longer\nsubstrates such as (POG) .\n“Nevertheless, ExoLead-driven unwinding of (POG) in the complex may be restricted by steric hindrance\n10\nbetween the PKD1 of ColHMT-N and CM of ColHMT-C already bound at the C-terminus (Fig. 3a and\nSupplementary Fig. 19). This steric hindrance could impede N-terminal progression of ColHMT-N and thereby\nlimit further N-terminal unravelling of (POG) , potentially reducing stable engagement of the partially\n10\nunwound middle and trailing strands by ExoMiddle and the active site, respectively.”\nLines 200-201: Concerning the observed complete unfolding of (POG)₁₂ in complex with ColHMT, the authors\nshould clarify why this observation supports the conclusion of N-to-C directional movement along the triple\nhelix. Additionally, please explain why a C-to-N directional movement would be inconsistent with the data, as\nthis is not immediately clear from the text. Please also discuss why not a similar behavior (i.e. complete\nunwinding of the triple helix upstream of the C-terminally bound ColH) was observed regarding the complex\nwith (POG)10.\nResponse: We appreciate the comment regarding the ColHMT–(POG) complex. Assuming that regions that are\n12\neasier to unwind are preferentially recognised by ColH, we initially hypothesised that the second ColHMT\nmolecule would unwind and stabilise the three triplets at the N-terminus of (POG) , resulting in a 2:1 ColHMT–\n12\n(POG) complex such as a 2:1 ColHMT-(POG) complex. However, our experimental observations did not\n12 10\nsupport this scenario.\nAs described in the manuscript, even if ColHMT were to bind to both termini of (POG) and unwind three triplets\n12\nat each end, the central region (about six triplets) would still retain the triple-helical structure. Therefore, a\npentameric dumbbell-like structure would be expected (Fig. 4f, middle). However, such a structure was not\nobserved. This can be reasonably explained by ColHMT bound at the N-terminus migrating to the C-terminus\nwhile progressively unwinding the triple helix (Fig. 4f, right).\nSince ColHMT can bind to any site on (POG) and translocate bidirectionally along the triple helix, as noted by\n12\nthe reviewer, we cannot exclude an alternative possibility in which a second ColHMT molecule binds\nimmediately adjacent to ColHMT-C and induces global unwinding of (POG) by migrating from the C- to N-\n12\nterminus. To address this issue, we have revised the Results section to explicitly clarify these possibilities (Lines\n282–283).\nRevised (Lines 282–283):\n“Here, we cannot exclude that a second ColHMT binds adjacent to ColHMT-C and subsequently migrates toward\nthe N-terminus, inducing global unwinding of (POG)₁₂.”\nLine 203: “shows [a] dynamic “closed” state]” Please correct.\nResponse: Thank you for this note. The text has been corrected to read “shows a dynamic ‘closed’ state” in the\nrevised manuscript.\nLine 204/Line 206: The authors correctly note that their findings “support” (line 104) the conclusion that ColH\ncan adopt a ratcheted state only when bound to a partially unwound substrate. In light of this, the subsequent\nstatement that “[t]hese results demonstrate that ColH exploits …” should be moderated, as the data support\nthe claim but do not entirely demonstrate it.\nResponse: Thank you for raising this point. Accordingly, we have revised the wording in Lines 204–206 of the\noriginal manuscript by replacing ‘demonstrate’ with ‘suggest,’ and have revised the sentence to more accurately\nreflect the scope of our findings.\nLine 207: Please replace “unlaying” with “unwinding”.\nResponse: The word ‘unlaying’ was chosen because the three strands are unwound and fixed in three different\nspatial positions.\nLine 211: The manuscript mentions “previous studies”; please include the appropriate citations to clarify which\nstudies are being referenced.\nResponse: We acknowledge the lack of clarity. We have now added the appropriate citations to identify the\nstudies being referenced.\nLine 212: Please change to plural, i.e. “how bacterial collagenases recognize the collagen triple helix and\nexploit its geometry for processive stand-specific cleavage.\nResponse: We appreciate the comment. We have revised the text to use the appropriate form.\nDiscussion: Please elaborate on how the presented results fit with the observed “open” conformation of\nbacterial collagenases reported by Eckhard et al (Ref. 3), Wang et al. (Ref.10), Ikeuchi et al. (Ref.15), designated\nin this manuscript as “wide-open” conformation. It would be very interesting to see a figure in which the\ndimensions of the different opening or closing states, respectively, are compared.\nResponse: Thank you for this valuable comment. In the revised manuscript, we included Supplementary Fig.\n21 and text comparing the dimensions of the different opening and closing states, showing the closed\nconformation of ColH relative to the previously reported ‘wide-open’ conformations of other bacterial\ncollagenases (refs. 5, 7, 15 in the revised manuscript). This comparison highlights how the distinct open–closed\ndynamics may relate to differences in substrate specificity.\nRevised (Lines 315–320):\n“These residues may also help ColH recognise vulnerable sites in tropocollagen as it slides from the N- to C-\nterminus (Fig. 5c and d). Notably, the dynamic ‘closed’ conformation of ColH is in stark contrast to that of\nhomologous enzymes such as ColG and some members of the M9A subfamily, which adopt more widely opened\nAD–PD arrangements (Supplementary Fig. 21)5, 7, 15. The closed-biased architecture of ColH may enhance its\nability to capture rare, thermally populated local fluctuations of the collagen triple helix, thereby promoting\nactive unwinding.”\nSupplementary Fig. 21. Structural comparison of the CM of M9 family collagenases. a Overall structure\nof M9B collagenase ColG from H. histolytica (PDB ID: 2Y50). b Overall structure of M9A collagenase VhaC\nfrom V. harveyi (PDB ID: 7ESI). c Overall structure of M9A collagenase Ghcol from G. hollisae (PDB ID:\n7WSS). In each case, the superposition with ColHWT from H. histolytica (this study) was performed using the\nPD catalytic subdomain, shown in the bottom panel.\nLine 218: The ColHMT-N-POG10 complex represents an “unproductive” substrate binding mode (resulting\nprobably from the mis-staggered triple helix at this location). While this is unfortunate, this complex nonetheless\nillustrates how ColH could utilize the captured, but unwound substrate as “rail tracks” for its one-dimensional\ndiffusion, reminiscent of DNA-binding clamps. Please change the text to reflect this conclusion by the authors.\nResponse: Thank you for this valuable comment. We have revised the text to clarify that, although the ColHMT-\nN-(POG) complex represents an ‘unproductive’ substrate-binding mode (likely due to the mis-staggered triple\n10\nhelix at this location and/or the steric hindrance by ColHMT-C), it nonetheless illustrates how ColH could use\nthe captured, but unwound, substrate as ‘rail tracks’ for one-dimensional diffusion, analogous to DNA-binding\nclamps.\nRevised (Lines 301–306):\n“As shown in the ColHMT–N–(POG) complex, which represents an unproductive binding mode likely arising\n10\nfrom a mis-staggered triple helix and/or steric hindrance by ColHMT-C, the CM appears to use the captured\nsubstrate as ‘rail tracks’ for one-dimensional diffusion. This directional movement may enable the enzyme to\nsearch for vulnerable regions, reminiscent of the behaviour of DNA-binding clamps and endonucleases (Figs.\n3 and 5c, d)25, 26.”\nLine 235-236: I would recommend the use of [cleavage] instead of [release]: “This captured bight is critical\nfor translocation and reloading of the substrate, enabling [cleavage] of a Gly-X-Y tripeptide”.\nResponse: Thank you for this helpful suggestion. We have replaced ‘release’ with ‘next-step cleavage.’\nLine 238: “underscoring [the] mechanistic divergence between bacterial and mammalian enzymes.” Please\ncorrect.\nResponse: Thank you for this note. We have corrected the sentence to read “underscoring the mechanistic\ndivergence between bacterial and mammalian enzymes.”\nLines 240-248: Please clarify that the authors present and propose here a new refined model of collagen\ndegradation by bacterial collagenases.\nResponse: Thank you for this constructive comment. We have rewritten the text to explicitly present the refined\nmodel of collagen degradation by bacterial collagenases.\nRevised (Lines 332–343):\n“The cleavage mode also differs between bacterial collagenases and MMPs. MMPs act exclusively as\nendopeptidases, whereas ColH advances toward the N-terminus by releasing trailing-strand tripeptides through\ncarboxytripeptidase activity13. Here, we present a refined model in which, after each cleavage event, the three-\nresidue bight passively dissociates from the active-site cleft due to reduced post-cleavage affinity. The next\ntriplet then moves into the S1′–S3′ subsites as the CM domains realign (Fig. 5g, h). This requires transient\nloosening of AD–PD interactions, allowing the AD to twist back by ~10 Å (one triplet) (Fig. 2d). A conserved\n‘wall motif’ in the distal S3′ pocket, unique to M9B collagenases, enforces this stepwise C-to-N progression.\nIntermittent AD engagement with the leading and middle strands further ensures triplet-spaced translocation.\nThrough cycles of ‘closed’ and ‘ratcheted’ states, ColH trims one trailing-strand triplet at a time, consistent with\nthe C-to-N directionality previously observed for ColG at the fibril level14.”\nLine 242: The term “unloads” implies active intervention by the collagenase. Was this the intended meaning?\nIt seems more likely that the cleaved products dissociate due to reduced affinity for the active-site cleft.\nResponse: We appreciate the helpful comment. We revised the text to indicate that the cleaved products likely\ndissociate due to reduced affinity for the active-site cleft, rather than being actively ‘unloaded’ by the enzyme.\nRevised (Lines 334–337):\n“Here, we present a refined model in which, after each cleavage event, the three-residue bight passively\ndissociates from the active-site cleft due to reduced post-cleavage affinity. The next triplet then moves into the\nS1′–S3′ subsites as the CM domains realign (Fig. 5g, h).”\nLine 246: I suggest including a reference to Watanabe-Nakayama et al. (2016), who demonstrated catalytically\nproductive unidirectional movement of ColG on collagen at the fibril level from C-to-N terminus, as this would\nprovide useful context for the reader.\nResponse: Thank you for this suggestion. We have added a reference for Watanabe-Nakayama et al. (2016) in\nLine 343 to provide context regarding the catalytically productive unidirectional movement of ColG on collagen\nfrom the C- to N-terminus at the fibril level.\nWhere is Reference 26 cited?\nResponse: Thank you for noting the missing citation. We have removed ref. 26 in the revised manuscript.\nLine 250 (typo->350): “was added [to remove the GST tag]”\nResponse: We revised the manuscript ss suggested.\nLine 361: Please provide buffer/elution details regarding the ion-exchange chromatography.\nResponse: Following your guidance, we made the following revisions:\nRevised (Lines 404–408):\n“After removal of the GST tag by affinity chromatography, proteins were purified by ion-exchange\nchromatography with Q-Sepharose Fast Flow beads (Cytiva) equilibrated with 50 mM Tris-HCl (pH 7.5)\ncontaining 1 mM CaCl . The flow-through fraction containing ColHWT CM was further purified by size-\n2\nexclusion chromatography as described above.”\nLine 368: Please provide buffer details for the reconstitution of the peptide solutions.\nResponse: Thank you for pointing this out. The peptides were reconstituted in the same cryo-EM buffer used\nthroughout this study (50 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1 mM CaCl₂). This information has been added\nto the Methods section (Lines 415-416) to improve clarity.\nLine 672: It would be improve clarity if the authors elaborated why the (hypothesized) N-terminally bound ColH\nmolecule could not be observed by cryo-EM, while the C-terminally bound ColH was.\nResponse: As shown in Fig. 4, in the case of (POG) , stable binding of the second ColHMT molecule (ColHMT-\nthereby promoting overall unwinding. Once fully unwound, the two molecules (ColHMT-N and ColHMT-C) are\ndifficult to analyse collectively owing to their orientational flexibility or the possible dissociation of one\nmolecule from the unwound peptides. Since ColHMT-N and ColHMT-C are expected to recognise the unwound\npeptide segments similarly, only an averaged structure is obtainable.\nWe have revised the text (Lines 283–285) to incorporate this point.\nRevised (Lines 283–285):\n“Once fully unwound, the two molecules are difficult to analyse collectively owing to their orientational\nflexibility or possible dissociation of one molecule from the unwound peptides.”\nLine 507: The method used to assess collagenolytic activity may not be perfectly able to distinguish between\ngelatinolytic and collagenolytic activity, as the assay was performed at 37°C, a temperature at which singly-\naccessible collagen molecules on the surface of the fibril will already start to denature (doi:\n10.1073/pnas.032307099).\nResponse: Thank you for this valuable comment. It is well known that triple-helical tropocollagen is hydrolysed\nby true collagenases, but that denatured collagen (gelatin) is hydrolysed by a wide variety of proteases.\nWang et al. (Nat. Commun. 13, 566, 2022) reported that trypsin (negative control) showed negligible activity against\ninsoluble type I collagen fibres, suggesting that the amount of gelatin on the surface of collagen fibres does not\nsignificantly affect the collagenase assay. Apart from this, the amount of soluble product in the buffer control\nwas ~1.5% of that in the positive control (ColHWT), showing minimal spontaneous release of the water-soluble\nsubstrate.\nRegarding the reproducibility of the data, although being only a minor issue, I would like to remark that I could\nnumerically not reproduce the RMSD deviations for bond lengths and bond angles for all reported EM structures\nusing PHENIX. Please check.\nResponse: Thank you for carefully checking the stereochemical parameters. We think that the small\ndiscrepancies in bond‑length and bond‑angle RMSD values likely arise from differences in PHENIX versions\nand refinement settings. In our study, model refinement was performed using PHENIX version 1.20.1-4487\nwith default geometry restraints optimised for cryo‑EM structures.\nDetails of the software versions used in the structural analysis are described in the Nature Portfolio Reporting\nSummary and are also available on the PDB website as part of the registration information for each X-ray or\ncryo-EM structure.\nRegarding the reported crystal structure for ColH CM, the Rwork/Rfree ratio (0.224/0.283) indicates overfitting\nof the density. Please revise your model.\nResponse: As noted, the R value differed by ~6% from the R value in the original model, raising concerns\nfree work\nabout overfitting. We therefore re-refined the structure, with particular attention to the placement of water\nmolecules and the AD domain, which exhibited elevated B factors. Consequently, we obtained an improved\nmodel with R = 0.231 and R = 0.271, which has been deposited in the Protein Data Bank under accession\nwork free\ncode 9CME. Accordingly, we have revised Supplementary Table 2.\nLine 709:”as described in Extended Data Fig. 6, and Supplementary Videos 1 and 2”, these references appear\nto be mis-placed.\nResponse: Thank you for this note. We have corrected the legend for Supplementary Fig. 1.\nLine 710: Please correct to “activator domain” (not accessory domain).\nResponse: Thank you for noting this mistake. We have corrected ‘accessory domain’ to ‘activator domain’ in\nthe legend for Supplementary Fig. 1.\nLine 711: Remove full stop after ColHWT.\nResponse: Thank you for this note. We have removed the full stop after ‘ColHWT’ in the legend for\nSupplementary Fig. 1.\nLine 741: “as described in Extended Data Fig. 5 and [illustrated] in Supplementary Video 3”\nResponse: Thank you for your careful review. This part of the sentence has been revised to read “as described\nin Supplementary Fig. 5 and illustrated in Supplementary Video 3.”\nLine820: Please correct to “PPG10”.\nResponse: Thank you for pointing this out. ‘(PPG) ’ has been used throughout the revised manuscript.\nmap) and the corresponding resolution.\nResponse: To address this comment, the composite map and its associated composite half maps generated by\nphenix.combine_focused_maps were imported into cryoSPARC, and map-to-map FSC curves were computed\nusing the Validation (FSC) job.\nIn the revised manuscript, the resulting curves and corresponding resolution values conforming with the FSC =\n0.143 criterion have been added to Supplementary Figs 1, 8, 9, and 20.\nPoint-by-point responses for reviewer’s comments\nReviewer #1 (Remarks to the Author)\nI appreciate the authors efforts to address all critiques raised in the first round of review. From this reviewers\nperspective the manuscript and models have been significantly improved. I support publication of this manuscript.\nWe are grateful for your careful assessment of our manuscript and for the substantial time and effort you have invested\nin the review process. We greatly appreciate your acknowledgment of the improvements made in the revised\nmanuscript, and are encouraged by your support for publication.\nReviewer #2 (Remarks to the Author)\nThe authors have made good efforts to address the reviewer comments and the manuscript has significantly improved.\nSome minor suggestions for the revised manuscript are as follows:\nWe thank the reviewer for the positive and encouraging assessment of our revised manuscript and are also grateful\nfor the additional minor suggestions, which have helped us further refine the quality of the manuscript. Our detailed\nresponses to each comment are provided below.\n1. The author did not include an introduction to the collagenases of the two subfamilies (M9A and M9B) of the M9\nfamily in the Introduction, which made the analysis and discussion of M9A and M9B collagenases in the Results and\nDiscussion sections appear somewhat abrupt. Therefore, I suggest adding an introduction to M9A and M9B\ncollagenases between lines 55 and 63, such as domain composition, characteristics of cleavage sites, structural\nfeatures, etc.\nResponse: We thank the reviewer for this helpful suggestion. In response, we have revised the Introduction (lines\n55–69) to include a brief contextual overview of the M9 family and its subdivision into the M9A and M9B subfamilies.\nThis addition clarifies their placement within the broader protease classification framework and provides smoother\ncontinuity with the analysis presented in the Results and Discussion. Detailed descriptions of domain composition,\ncleavage characteristics, and structural features remain available in the cited literature.\nRevised (Lines 55–69):\n“To degrade collagen-based structures, bacteria and metazoa produce collagenases, i.e. zinc-dependent\nmetallopeptidases belonging to clan MA in the MEROPS database4. Bacterial collagenases belong to the M9 family\n(gluzincins) based on the sequence similarity5, 6. M9A collagenases are produced mainly by marine bacteria such as\nspecies of Vibrio and Grimontia, whereas M9B collagenases are produced by terrestrial bacteria such as species of\nClostridium and Bacillus. Hathewaya histolytica (formerly Clostridium histolyticum) produces two classes of M9B\ncollagenases (ColG and ColH) 7-9 that act as virulence factors in soft-tissue infections, which have also been adapted\nfor clinical applications, including wound debridement, fibrosis treatment, and islet isolation10. Bacterial collagenases\ncontain multiple domains, including an activator domain (AD) and thermolysin-like peptidase domain (PD), which\ncooperate to unwind and cleave fibrillar collagens11. PD-mediated cleavage depends on prior AD-mediated\nunwinding12, 13. However, how the enzyme unwinds the triple helix and maintains the unwound state while engaging\nall three strands remains unclear.\nMammalian matrix metalloproteinases [MMPs, M10 family (metzincins)] have been widely studied and play a tightly\nregulated role in ECM remodelling.”\nAdded references:\n4Rawlings, N. D. et al. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a\ncomparison with peptidases in the PANTHER database. Nucleic Acids Res. 46, D624-D632 (2018).\n5Zhang, Y. Z., Ran, L. Y., Li, C. Y. & Chen, X.L. Diversity, structures, and collagen-degrading mechanisms of\nbacterial collagenolytic proteases. Appl. Environ. Microbiol. 81, 6098-6107 (2015).\n6Duarte, A. S., Correia, A. & Esteves, A. C. Bacterial collagenases - A review. Crit. Rev. Microbiol. 42, 106-126\n(2016).\n7Mookhtiar, K. A. & Van Wart, H. E. Clostridium histolyticum collagenases: a new look at some old enzymes. Matrix\nSuppl. 1, 116-126 (1992).\n8Yoshihara, K., Matsushita, O., Minami, J. & Okabe, A. Cloning and nucleotide sequence analysis of the colH gene\nfrom Clostridium histolyticum encoding a collagenase and a gelatinase. J. Bacteriol. 176, 6489-6496 (1994).\n9Matsushita, O. et al. Gene duplication and multiplicity of collagenases in Clostridium histolyticum. J. Bacteriol. 181,\n923-933 (1999).\n2. L70-L72: The author compares the differences in cleavage characteristics of collagen peptide chain between M9\nbacterial collagenases and M10 mammalian matrix metalloproteinases here. However, merely citing the description\nof M9B collagenase is insufficient (ref 13), as M9A collagenases also exhibit cleavage site specificity for Y-Gly bonds\nin collagen peptide chains releasing collagen tripeptide Gly–X–Y (J Agric Food Chem. 2025 73:22079-22088; Nat\nCommun. 2022 13:566; Appl Environ Microbiol. 2022 88:e0167721). There is also a similar deficiency in lines 181\nto 182.\nResponse: We thank the reviewer for this helpful comment. For M9A (Vibrio) collagenases, carboxytripeptidase\nactivity has been inferred from collagen fibril hydrolysis experiments showing early accumulation of tripeptide\nproducts (Ikeuchi et al., J. Biol. Chem. 2022). Furthermore, the Yaa–Gly bond specificity has been shown by structural\nand high-resolution LC–MS studies (Wang et al., Nat. Commun., 2022; Zhao et al., J. Agric. Food Chem. 2025; Wang\net al., Appl. Environ. Microbiol., 2022). We thank the reviewer for drawing our attention to these important studies.\nWe have revised the manuscript accordingly and have added citations to the above-mentioned references (Lines 75–\n80).\nRevised (Lines 75-80):\n“Unlike MMPs, which act exclusively as endopeptidases, bacterial collagenases exhibit both endopeptidase and\ncarboxytripeptidase activities, preferentially cleaving Y–Gly bonds12, 19-22. The carboxytripeptidase processively\ntrims Gly–X–Y triplets from the initial site of engagement towards the N-terminus. High-speed atomic force\nmicroscopy has demonstrated catalysis-coupled movement of a clostridial collagenase in the N-terminal direction23,\nyet how strand engagement supports this processive advancement remains unclear.”\nAdded references:\n20Wang, Y. et al. Mechanistic insight into the fragmentation of type I collagen fibers into peptides and amino acids\nby a Vibrio collagenase. Appl. Environ. Microbiol. 88, e0167721 (2022).\n21Ikeuchi, T. et al. Crystal structure of Grimontia hollisae collagenase provides insights into its novel substrate\nspecificity toward collagen. J. Biol. Chem. 298, 102109 (2022).\n22Zhao, W. X. et al. Mechanistic insight into the cleavage site specificity of Vibrio collagenase VhaC to the Y-G bonds\nin collagen. J. Agric. Food Chem. 73, 22079-22088 (2025).\n3. I suggest adding orange and purple color blocks in the blank area of Fig. 2f to represent the substrates Pz-peptide\nand collagen, respectively, so that readers can quickly grasp the activity information of the wild-type protein and its\nmutants toward the two substrates. This does not seem to contradict the suggestion from Reviewer 3.\nResponse: We thank the reviewer for his/her helpful suggestion. We have placed orange and purple color blocks at\nthe top of blank space of Fig. 2f, while leaving the relevant caption unchanged as previously advised by Reviewer 3.\nAdditionally, in response to the editor’s requests, we have added blue dots representing the mean values of each\ntriplicate to the figure and have included an explanation in the figure caption.\nFig. 2 ColH binds to unwound C-terminus of (POG) . f Relative Pz-peptidase (orange) and collagenase (purple)\n10\nactivities of wild-type and mutant ColH. All assays were performed in triplicate across three independent\nexperiments. Data are presented as mean values ± SEM. Blue dots represent the mean values of each triplicate. The\nstatistical differences between wild-type and mutant ColH were examined by a two-tailed t-test. The exact p values\nfor Pz-peptidase and collagenase activities were 0.574 and 1.28 × 10-3 for ExoLead, 9.26 × 10-3 and 2.05 × 10-3 for\nExoMiddle, 0.488 and 1.07 × 10-5 for F90A, 0.408 and 9.34 × 10-3 for Y235A, 4.83 × 10-5 and 1.15 × 10-6 for W431A,\nand 1.09 × 10-4 and 1.19 × 10-3 for E416Q, respectively; *, p < 0.01; ns, non-significant (p ≥ 0.01). Source data are\nprovided.\nReviewer #3 (Remarks to the Author)\nThe quality and clarity of the revised manuscript have been significantly enhanced. The concerns raised in my\nprevious comments have been thoroughly addressed, and I appreciate the considerate and detailed responses to my\ncomments.\nUpon review of the revised manuscript, four minor issues have arisen that need to be addressed prior to publication:\nWe are grateful for your thorough assessment of our revised manuscript and for the considerable time and effort you\nhave devoted to evaluating our work. Your considerate and detailed comments have substantially contributed to\nenhancing the rigor and presentation of the manuscript. We have carefully considered the four remaining minor issues\nand provide our detailed responses below.\n- Line 185: Please correct: “This distortion is critical for Zn2+-mediated polarization of the scissile carbonyl bond”\ninstead of “chelation”. The catalytic zinc ion functions as a Lewis acid in metallopeptidases.\nResponse: We appreciate the reviewer for giving us the opportunity to reconsider the wording ‘chelation.’ As its\netymology suggests, ‘chelation’ (or ‘chelate’) refers to a structural arrangement in which a ligand binds a metal ion\nthrough multiple coordination points, resembling a crab grasping an object with its claws. Therefore, this term is not\nappropriate for describing the interaction between Zn²⁺ and a single scissile carbonyl oxygen.\nInstead, we use the term ‘coordination’ to describe the structural arrangement in which the distorted scissile peptide\nbond is properly positioned relative to the Zn²⁺ ion. This coordination ensures precise alignment of the carbonyl\noxygen with the catalytic metal centre.\nFollowing correct coordination of the scissile carbonyl oxygen, the Zn²⁺ ion functions as a Lewis acid, accepting\nelectron density from the oxygen atom and thereby enhancing polarisation of the carbonyl group. This polarisation\nincreases the partial positive charge on the carbonyl carbon, which is essential for initiating nucleophilic attack by\nthe activated water molecule (OH⁻). We have added a brief description of this polarisation step in a separate paragraph\nwithin the catalytic centre section.\nRevised (Lines 191-192):\n“This distortion is critical for Zn²⁺ coordination of the scissile carbonyl oxygen.”\nRevised (Lines 230-232:\n“At the catalytic centre, the scissile peptide carbonyl oxygen coordinated the Zn²⁺ ion, increasing polarisation of the\ncarbonyl bond and priming it for nucleophilic attack. No ordered water molecules were observed near either the\nscissile bond or residue Gln416.”\n- The authors state in their response to the comment on former Lines 200–201 that ColH can translocate\nbidirectionally along the triple helix. To ensure consistency with this clarification, it would be appropriate to modify\nthe wording in Lines 315–316 to “as it slides along the triple helix” instead of “from the N- to C-terminus.”\nResponse: We thank the reviewer for this valuable suggestion. We have revised Lines 315–316 as recommended to\nensure consistency in the description.\nRevised (Lines 321-322):\n“These residues may also help ColH recognise vulnerable sites in tropocollagen as it slides along the triple helix (Fig.\n5c and d).”\n- Supplementary Fig.16. Figure caption: Please provide the used PDB entry codes.\nResponse: We thank the reviewer for this valuable comment. The PDB entry codes have now been incorporated into\nthe caption of Supplementary Fig. 16. The revised caption reads as follows:\nRevised:\nSupplementary Fig. 16 Ligand-induced contraction of the PD catalytic subdomain in M9B collagenases.\nSuperposition of PD structures from (POG)₁₀-bound ColHMT (PDB ID: 9LQJ, this study) and substrate-free ColHWT\n(PDB ID: 9LRK, this study) (top); isoamylphosphonyl-Gly-Pro-Ala-bound (PDB ID: 2Y6I) and inhibitor-free (PDB\nID: 2Y50) ColG (bottom left); and isoamylphosphonyl-Gly-Pro-Ala-bound (PDB ID: 4AR8) and inhibitor-free (PDB\nID: 4AR9) ColT from Clostridium tetani (bottom right). In each comparison, superposition was performed using the\nN-terminal half-domain. Contraction of the C-terminal half-domain relative to the well-aligned N-terminal half-\ndomain is indicated by arrows. The N-terminal and C-terminal half-domains are coloured grey and cyan, respectively,\nand the helper subdomain is coloured blue.\n- Supplementary Fig.18: Please rephrase the figure caption to reflect that the presented model structure is the result\nof homology modelling using MOE with complex ColHMT-(POG)10 (PDB: 9LQJ) as initial template.\nResponse: We have revised the caption of Supplementary Fig. 18 to clearly state that the presented model structure\nwas generated in silico based on PDB entry 9LQJ. We modified the figure caption as follows.\nRevised:\nSupplementary Fig. 18. Model structure of ColHWT–Pz-peptide complex.\nThe structural model of ColHWT–Pz-peptide complex was generated in silico by reverting the single mutated residue\nto wild type and replacing the bound (POG) peptide with the Pz-peptide in the ColHMT–(POG) structure (PDB\n10 10\nID: 9LQJ), followed by energy minimisation in Molecular Operating Environment. a Overall structure. b Enlarged\nview of active site in the complex. Pz-peptide bound to PD coordinates the zinc ion via the carbonyl oxygen of Leu2\n(P1), and the adjacent Pro1 (P2) forms the CH–π and hydrophobic interactions with Trp431. The orange columns\nrepresent the carbon atoms of Pz-peptide."}]

Decision letter