Structure and Mechanism of Bacterial Collagenases

Collagen is the most abundant and, at the same time, one of the most robust proteins in mammals. Degradation and remodeling of collagen is implicated in many physiological, but also pathological situations, and is accomplished only by specially adapted enzymes, collagenases. Collagen cleavage appears to occur in a complex two-step process that involves local unwinding of the collagen triple helix followed by peptide bond hydrolysis of the separated polypeptide chains. Vertebrate collagenases belong to the well studied family of MMPs, for which crystal structure information and detailed mechanistic data are available. By contrast, very little structural information about bacterial collagenases, which evolved independently from mammalian collagenases, is available. Collagenases from clostridia have been studied for decades, reflecting their broad spectrum of biotechnological applications. For pathogenic clostridial strains such as C. tetani or C. histolyticum, additional interest is directed to targeting these clostridial collagenases for drug discovery. We propose to determine the crystal structure and function of three related clostridial collagenases from C. histolyticum and C. tetani. These three enzymes differ in important details of their domain composition and are, therefore, ideal candidates to deduce the biological role of the individual domains and how these domains concert in the catalyzing collagen degradation.

Collagen is the by far most abundant protein in humans. Consisting of three twisted peptide chains, its architecture results in the characteristic collagen triple-helix. It is this particular architecture that confers the strength and stability to collagen as found in cartilage, bone or tendon. At the same time, the triple-helical architecture makes the remodeling or degradation of collagen particularly demanding and can be only accomplished by highly specialized enzymes, collagenases. Collagenases are found in the human body for natural collagen remodeling; however, the most efficient collagenases are produced by certain bacteria, in particular Clostridia. The exact mechanism of collagen degradation was unclear.During the FWF - funded project we could determine the crystal structure of several clostridial collagenases and decipher the fundamentals of the enzymatic mechanism of collagen degradation.The collagenase has a pincer-like architecture. The reactive center is completely contained in one jaw. However, the second jaw is essential for the breakdown of collagen. The structure analysis revealed that the two jaws cooperate in for unwinding the triple-helical collagen molecule, and in a second step to cleave the untwisted, individual peptide chains. The accompanying domain movement can be entropically driven by dehydration of the collagen molecule. This two-step reaction mechanism is supported by mutagenesis studies. The activity at the catalytic site depends on two metals, the catalytic zinc and a close by calcium ion. Both metals are exploited as regulatory elements.These structural and enzymatic works support a wide range of biotechnological and pharmaceutical applications. In fact, collagenases are already being used in the treatment of diseases of the connective tissue of the palm ( Dupuytren's contracture ) or for islet cell isolation used for the treatment of diabetes.