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.

Anchoring fibrils formed by collagen VII play a critical role in stabilizing the dermal-epidermal junction. The N-terminal non-collagenous (NC1) domain of collagen VII binds firmly to basement membrane components including collagen IV and has also been reported to interact with mesenchymal fibrillar collagens via its von Willebrand factor A-like domain 2 (A2 domain). To elucidate how collagen VII recognizes fibrillar collagen, we performed yeast two-hybrid screening using a triple-helical random peptide library, which resulted in the identification of a Met-Gly-Φ (Φ ; aromatic amino acid residue) motif. Biochemical analysis with synthetic triple-helical peptides revealed a binding preference of Trp > Phe as the Φ residue by the A2 domain despite Trp being absent in native collagens. The crystal structure of the A2 domain in complex with the Nle (Met surrogate)-Gly-Trp-containing peptide revealed a unique mechanism by which two distinct hydrophobic pockets of the A2 domain accommodate the Nle and Trp residues corresponding to the Met-Gly-Φ motif, engaging all three chains of the triple helix. Subsequent molecular dynamics simulations demonstrated that the A2 domain recognizes the corresponding native Met-Gly-Phe motif in a similar manner, but with lower affinity, implying a transient interaction with mesenchymal collagens. The findings obtained in this work suggest models in which transient A2-triple helix interaction promotes the recruitment of collagen I and III fibrils into the arc-shaped structure of anchoring fibrils. This also provides a foundation for linking structural understanding to skin fragility diseases caused by collagen VII dysfunction.

Integrins α1β1, α2β1, α10β1, and α11β1 serve as primary collagen receptors, recognizing the GxxGEx motif of the collagen triple helix via their αI domains. The Glu residue of the motif coordinates with a divalent metal cation at the metal ion-dependent adhesion site of the αI domain. Owing to this conserved recognition mechanism, previously identified binding sequences show low subtype selectivity. In this study, we identified triple-helical peptide ligands capable of selectively binding to the α2I domain in a manner independent of both Glu and a divalent metal cation. By employing yeast-two hybrid screening using a randomized triple-helical peptide library, we identified sequences in which canonical Glu was substituted by aliphatic residues such as Met. X-ray crystallographic analysis of the complex formed by the α2I domain and a triple-helical peptide containing GFOGMR (O: 4-hydroxyproline)—a Met-substituted variant of the canonical integrin-binding sequence GFOGER—revealed a novel binding mode. In this mode, the peptide was recognized by the closed form of the α2I domain in a metal cation-independent manner. Furthermore, this interaction mode enabled Met-containing peptides to specifically bind to the integrin α2I domain. These findings deepen our understanding of collagen recognition by collagen-binding integrins and provide insight for the discovery of ligands capable of selectively targeting collagen receptor-type integrins.

ABSTRACT

The fibrous structures of collagen provide physical strength and stability to tissues and organs. Abnormalities in their orientation, growth, and remodeling cause morphogenetic defects and serious diseases including fibrosis, so it is important to clarify how collagen fibers are correctly oriented and grown within tissues. However, this mechanism remains elusive, as few methods have been available to fluorescently stain collagen fibers with a simple protocol and to observe their structure in three dimensions. Here we present a facile method that enables fluorescent staining of collagen fibers in vertebrate tissues. In our method using DAF-FM, known as a NO detection probe, premature collagen fibers can be visualized via covalent binding to the allysine residues serving as precursors of cross-linking structures of collagen. In addition, we showed that the labeling method using two fluorescent probes with different colors, DAF-FM and DAR-4M, allows for pulse-chase observation of newly synthesized collagen fibers. Our method will be a breakthrough technique in future collagen studies.