Mechanism of bacterial collagenolysis

Collagens are the single most abundant proteins in mammals. They account for nearly 25% of the total body protein. Collagens are highly resistant to degradation and are essential for maintaining the integrity of tissues. Merely a very small number of mammalian enzymes is capable of collagen remodeling. Yet, several bacterial species from the genera Clostridium and Bacillus also have evolved collagenases. These bacterial collagenases enable them to utilize collagen for nutrition. Moreover, they provide pathogenic strains with a tool to facilitate host invasion, colonization, and toxin diffusion. Bacterial collagenases are zinc metalloproteases. They are highly efficient enzymes that can completely degrade collagen. Consequently, they have found widespread application in industry, research, and clinics. Yet, bacterial collagenases are not only interesting as a biotechnological tool. Given that clostridia encompass major human pathogens (such as Clostridium perfringens, C. histolyticum, C. tetani, and C. botulinum), and that the number of antibiotic resistances is growing, these clostridial collagenases represent also interesting targets for the development of a new class of anti-infective drugs. The mechanism by which bacterial collagenases can degrade collagen has remained a poorly understood. With the recent structure of the collagenase unit of ColG from C. histolyticum, we have accomplished a critical milestone for unraveling this puzzle. It allowed us to identify the two minimal functional units for collagen degradation and peptide cleavage in clostridial collagenases, the collagenase unit and the peptidase domain, respectively. Moreover, we were able to propose a two-step model of bacterial collagen degradation, in which the concerted opening and closing of the collagenase unit prime collagen triple helices for cleavage. This project aims at decoding the molecular mechanisms that govern collagen degradation by bacterial collagenases. For this purpose, we will target the minimal functional unit capable of collagen degradation, the collagenase unit, using X-ray crystallography and other biochemical and biophysical methods. We want to know how the collagenase unit can accomplish (i) collagen binding, (ii) unwinding of the collagen triple helix, and (iii) its cleavage, and we want to determine the role of interdomain dynamics in catalysis. This will allow us to gain an in-depth understanding of the mechanism of bacterial collagen degradation. Moreover, it will disclose routes to tune collagenase activity and it will allow us to custom-tailor bacterial collagenases for applications in industry, research, and medicine. In addition, it will guide the rational design of a new generation of highly specific collagenase inhibitors that could complement and replace existing antibiotic treatments.

Collagen is the by far most abundant protein in humans. It is very hard to degrade enzymatically. Only few enzymes can cleave it. One reason for this is that collagens are mostly found in large insoluble fibrillar assemblies in the body, from which a single soluble collagen molecule first has to be extracted for enzymatic processing. Then the enzyme has to unwind the individual triple-helical collagen molecule (i.e. tropocollagen), which is tightly intertwined like a thread, into its individual -chains. For cleavage of the -chains, the enzyme has to fit a single unwound -chain into its catalytic center. Yet, only few enzymes can accommodate the relatively rigid unwound collagen -chains in their active sites. Therefore, only a handful of specialized enzymes called collagenases exists that can degrade collagen, when it is properly folded and assembled into fibrillar structures, respectively. Very efficient collagenases are produced by bacteria. These collagenases are composed of an N-terminal collagenase unit, which adopts a pincer-shaped structure, and a varying composition of C-terminal polycystic disease-like domains (PKD) and collagen-binding domains (CBD). However, it is not well understood how the bacterial collagenases accomplish the complex task of collagen degradation. During the FWF - funded project we found that bacterial collagenases primarily bind to collagen in its different structural variants (fibrils, tropocollagen and gelatin) via their collagenase unit supported by their collagen binding domains, yet without the assistance of the PKD domain. We revealed that the collagenase unit needs to be flexible to cleave, but not to bind, insoluble collagen, possibly in order to be able to extract single collagen molecules from the fibrillar assembly. Surprisingly, we discovered using a newly developed tool for detection by circular dichroism that the task of unwinding soluble collagen does not require the action of both jaws of the collagenase unit, but that it is accomplished by one, the so-called activator domain. We could further identify individual residues in the activator domain critical for binding and unwinding of the substrate by mutagenesis studies. This activator domain-mediated unwinding mechanism clearly sets the bacterial collagenases apart from their mammalian counterparts. In addition, we used structural and enzymatic studies to develop and characterize inhibitors targeting bacterial collagenases, which could pave the way for the development of anti-virulence drugs against multidrug-resistant pathogenic bacteria. Besides this drug design aspect, our findings could also help to custom-tailor bacterial collagenases for distinct applications, as these enzymes are currently, for example, used for the treatment of diseases of the connective tissue (Dupuytren's contracture and Peyronie's disease) or for islet cell isolation used for the treatment of diabetes.