It has been postulated that triple-helical collagen is actively unwound by collagenases before peptide bond hydrolysis--a supposition that explains the small catalytic rate constant associated with collagenolysis. We propose an alternate model of collagen degradation that does not require active unwinding by collagenases, but instead suggests that the regions of collagen near the collagenase cleavage site can adopt either a native triple-helical or a partially unfolded conformation. In this model, collagenases preferentially bind to and stabilize partially unfolded conformers before cleaving the scissile bond. Existing experimental observations (which were previously taken to support active unwinding models) are reinterpreted using corroborative evidence from numerical simulations and found to be consistent with this framework. These data support the notion that collagen, like all other biological heteropolymers, undergoes thermal fluctuations that cause it to sample distinct structures in the neighborhood of the native state, and collagenolysis occurs when collagenases recognize the appropriate unwound conformers.

Excessive degradation of type I collagen is associated with a variety of human diseases such as arthritis, tumor metastasis, and atherosclerosis. Methods that further our understanding of collagenolysis may therefore provide insights into the mechanism of several important disorders. Prior experiments suggest that cleavage of collagen in vitro requires intact full-length collagenase, a multidomain protein containing both a catalytic and a hemopexin-like domain. In this work we demonstrate that type I collagen can be degraded at room temperature, a temperature well below the melting temperature of type I collagen, by collagenase deletion mutants that only contain the catalytic domain of the enzyme. Furthermore, these mutant enzymes hydrolyze the same peptide bond that is recognized by the corresponding full-length enzymes. Hence enzyme specificity at room temperature is achieved without the hemopexin-like domain. We demonstrate that these findings can be explained in light of a conformational selection mechanism that dictates that collagenases preferentially recognize and cleave preformed partially unfolded states of collagen.

Matrix metalloproteases (MMPs) cleave native collagen at a single site despite the fact that collagen contains more than one scissile bond that can, in principle, be cleaved. For peptide bond hydrolysis to occur at one specific site, MMPs must (1) localize to a region near the unique scissile bond, (2) bind residues at the catalytic site that form the scissile bond, and (3) hydrolyze the corresponding peptide bond. Prior studies suggest that for some types of collagen, binding of noncatalytic MMP domains to amino acid sequences in the vicinity of the true cleavage site facilitates the localization of collagenases. In the present study, our goal was to determine whether binding to the catalytic site also plays a role in determining MMP specificity. To investigate this, we computed the conformational free energy landscape of Type III collagen at each potential cleavage site. The free energy profiles suggest that although all potential cleavage sites sample unfolded states at relatively low temperatures, the true cleavage site samples structures that are complementary to the catalytic site. By contrast, potential cleavage sites that are not cleaved sample states that are relatively incompatible with the MMP active site. Furthermore, our findings point to a specific role for arginine residues in modulating the structural stability of collagen near the collagenase cleavage site. These data imply that locally unfolded potential cleavage sites in Type III collagen sample distinct unfolded ensembles, and that the region about the true collagenase cleavage site samples states that are most complementary to the MMP active site.

Collagen is a unique structural protein that imparts tensile strength to bone, tendons, and numerous other tissues. Like many biological polymers, collagen is continually synthesized and degraded in the extracellular space. While collagen degradation is a normal part of collagen homeostasis, excessive collagenolysis has been implicated in a number of human diseases such as arthritis, cancer, and atherosclerosis. In this work we demonstrate how molecular simulations can be used to study the mechanics of collagen degradation. Dynamical simulations, which model the structural fluctuations that collagen can undergo under physiologic conditions, reveal that portions of collagen are quite flexible-a somewhat counterintuitive finding. Moreover, this flexibility likely facilitates the recognition and cleavage of collagen by proteolytic enzymes. Experiments on collagenlike model compounds are consistent with these observations and demonstrate that new insights into the physical basis of collagenolysis can be obtained from a combination of experiment and computation. More importantly, these results highlight new avenues for the development of potential therapies for disorders that involve abnormal collagen catabolism.

domain. Myosin-7a unfolds at either high ionic strength or in the absence of ATP, revealing a clearly recognizable motor domain, the lever arm and some features of the tail region. C-terminal truncations were made to determine which portions of the multi-domained tail are necessary for the regulation. Removal of the last 99 amino acids which are highly conserved in all myosin-7a molecules and form a subdomain (termed MyTH7) of the FERM domain, or mutation of two conserved amino acids in this region, is sufficient to prevent folding of the molecule in the presence of ATP and activates the enzymatic activity. A construct consisting of the second FERM domain binds actin in an ATP-insensitive manner with a Kd of 30mM which is similar to the KATPase value for the full length molecule. We propose that at low actin concentration myosin-7a is folded and inactive, but at high actin concentrations such exists in actin bundles, it binds first via its tail binding site which then frees the motor domain to functionally interact with actin.