Structural insights into triple-helical collagen cleavage by matrix metalloproteinase 1

Abstract

Collagenases of the matrix metalloproteinase (MMP) family play major roles in morphogenesis, tissue repair, and human diseases, but how they recognize and cleave the collagen triple helix is not fully understood. Here, we report temperature-dependent binding of a catalytically inactive MMP-1 mutant (E200A) to collagen through the cooperative action of its catalytic and hemopexin domains. Contact between the two molecules was mapped by screening the Collagen Toolkit peptide library and by hydrogen/deuterium exchange. The crystal structure of MMP-1(E200A) bound to a triplehelical collagen peptide revealed extensive interactions of the 115-Å-long triple helix with both MMP-1 domains. An exosite in the hemopexin domain, which binds the leucine 10 residues C-terminal to the scissile bond, is critical for collagenolysis and represents a unique target for inhibitor development. The scissile bond is not correctly positioned for hydrolysis in the crystallized complex. A productive binding mode is readily modeled, without altering the MMP-1 structure or the exosite interactions, by axial rotation of the collagen homotrimer. Interdomain flexing of the enzyme and a localized excursion of the collagen chain closest to the active site, facilitated by thermal loosening of the substrate, may lead to the first transition state of collagenolysis. extracellular matrix | X-ray crystallography | protease T he interstitial collagens I, II, and III are the major structural proteins in connective tissues such as skin, bone, cartilage, tendon, and blood vessels (1). They consist of three α chains with repeating Gly-X-Y triplets (X and Y are often proline and hydroxyproline, respectively) that intertwine each other to form a triple helix of ∼300 nm in length (2). Interstitial collagens are resistant to most proteolytic enzymes, but vertebrate collagenases cleave them at a single site approximately three-quarters of the way from the N terminus of the triple helix, thus initiating collagenolysis (3). Owing to this unique activity, collagenases play important roles in embryo development, morphogenesis, tissue remodeling, wound healing, and human diseases, such as arthritis, cancer, and atherosclerosis (4, 5).Matrix metalloproteinase 1 (MMP-1) is a typical vertebrate collagenase (3). It consists of an N-terminal catalytic (Cat) domain containing an active-site zinc ion and a C-terminal hemopexin (Hpx) domain comprised of a four-bladed β-propeller, which are connected by a linker region (6, 7). Although the Cat domain can cleave a number of noncollagenous proteins including heat-denatured collagen (gelatin), its activity on native triplehelical collagen is negligible. The combination of the Cat and Hpx domains is required for MMP-1 to be able to degrade native collagen, and the same is true for all other collagenolytic MMPs, namely MMP-2, MMP-8, MMP-13, and MMP-14 (3). How collagenases interact with collagen and how the Hpx domain endows these enzymes with collagenolytic activity is not clearly understood. Another enigma of collagenolysis bec...

Enzymatic turnover of macromolecules generates long-lasting protein–water-coupled motions beyond reaction steady state

“…2B shows that the reaction with the triple-helical substrate induced an increase in fluorescence for 30 ms, which decayed exponentially until ∼400 ms, whereas the fluorescence after the reaction with the single-stranded substrate increased until 70 ms and then exponentially decreased until reaching steady state at ∼400 ms. These results are in line with previously reported analyses, suggesting that conformational changes of MT1-MMP take place during Michaelis complex formation for triple-helical collagen model peptides (24,25). The net change in tryptophan fluorescence (Fig.…”

“…degradation involves major conformational rearrangements of both enzyme and substrate to facilitate binding and catalysis (24,25). Therefore, we chose to study the role of solvation dynamics in more complex enzymatic reactions, such as the enzymatic degradation of collagen-and gelatin-like substrates by MT1-MMP, by our established X-ray-and THz-based integrated spectroscopic approach (Fig.…”

Definition of the Native and Denatured Type II Collagen Binding Site for Fibronectin Using a Recombinant Collagen System

“…Human collagenases, such as MMP-1 and MMP-13, cleave between Gly 775 and Leu 776 and divide collagen chains into 1 ⁄ 4 and 3 ⁄ 4 fragments (42,43). A recent study indicated that Leu residues at positions 776 and 785 were critical for MMP activity through direct interaction with the MMP catalytic and hemopexin domains, respectively (44). Because the minimum Fn binding sequence defined here overlaps with the key residues for MMP cleavage, MMP and Fn could compete for binding at this site.…”

Characterization of a Novel Subtilisin-like Protease Myroicolsin from Deep Sea Bacterium Myroides profundi D25 and Molecular Insight into Its Collagenolytic Mechanism

“…Collagenolytic proteases, such as MCP-01 (30, 42) and MMP-1, -8, and -13 (49,50), usually have a C-terminal domain that functions as a collagen-binding domain to mediate and facilitate collagen hydrolysis by the enzyme. However, there are also some collagenolytic proteases that do not need an additional domain for collagenolysis, such as MMP-12 (51) and the serine proteases from fiddler crab (16,18).…”