Structure and collagenolytic mechanism of a Vibrio collagenase

The collagenolytic mechanism of Vibrio collagenase, a virulence factor, remains unclear. Here, the authors report the structure of Vibrio collagenase VhaC and propose the mechanism for collagen recognition and degradation, providing new insight into bacterial collagenolysis.
Structure and collagenolytic mechanism of a Vibrio collagenase

Collagen constitutes up to 30% of the total proteins in mammals and is the major protein of the extracellular matrix (ECM). Of the 28 different types of collagens, type I fibrillar collagen is the most widespread and abundant, providing structural framework for skin, bones, tendons, and other connective tissues. The hierarchical structure of type I collagen is well organized. Tropocollagen, the smallest unit, is made up of three intertwined α-chains (two α1 and one α2 chains), each of which contains repeating Gly-X-Y triplets in the triple-helical region. The N- and C-telopeptides form the non-helical region of tropocollagen and involved in the formation of covalent cross-links (pyridinium compounds and pyrroles). Tropocollagen molecules are assembled into collagen fibrils via these covalent cross-links. Collagen fibrils further form into collagen fibers by interdigitation with proteoglycans in the ECM.

Collagenases that hydrolyze the triple-helical region of collagen under physiological context are found in animals and microorganisms. Vibrio collagenase is closely related to Vibrio pathogenesis for its role in native collagen degradation during host invasion, and is regarded as a virulence factor. However, information on the three-dimensional structure and the collagenolytic mechanism of Vibrio collagenase is still lacking. The aim of our study is to solve the structure of Vibrio collagenase and reveal its collagenolytic mechanism.  

 VhaC is a collagenase of Vibrio harveyi VHJR7. Sequence analysis indicated that VhaC is composed of a collagenase module (CM) containing a peptidase M9N domain (activator domain) and a peptidase M9 domain (peptidase domain), a polycystic kidney disease-like domain (PKD-like domain) and a bacterial prepeptidase C-terminal domain (PPC domain). We solved the crystal structure of the CM, which presents a saddle-shaped architecture. Small angle x-ray scattering (SAXS) studies showed that the side-by-side arrangement of the CM, the PKD-like domain, and the PPC domain results in a long and flat overall conformation of the full-length VhaC in solution. Several biochemical experiments demonstrated that the activator domain has the ability to bind triple-helical molecule and collagen fiber, but the peptidase domain has not. Therefore, the activator domain of VhaC is likely responsible for the initial collagen recognition before the peptidase domain comes into action. Further molecular dynamics studies and biochemical analysis indicated that the triple-helical collagen is initially docked in the activator domain of VhaC, and is close to the catalytic center of the peptidase domain for further hydrolysis along with the closing movement of CM. During the degradation of collagen fiber, the PKD-like domain functions as a linker between the upstream (CM) and downstream (PPC) domains, and the PPC domain function as a collagen-binding domain to anchor the enzyme molecules on the surface of collagen fiber. Based on our results, we proposed a model for the integrated collagenolytic mechanism of Vibrio collagenase VhaC. Our results provide new insight into the collagenolysis of Vibrio collagenases, which are helpful in the development of strategies to prevent and treat infection caused by pathogenic Vibrio species.