MMP-12 Catalytic Domain Recognizes Triple Helical Peptide Models of Collagen V with Exosites and High Activity

Abstract

Collagens are principal constituents of connective tissues. They are hydrolyzed during development, wound repair, and remodeling of the extracellular matrix. Dysregulated collagenolysis is active in inflammatory diseases such as atherosclerosis, cancer, rheumatoid arthritis, periodontitis, and liver and kidney pathologies. Matrix metalloproteinases (MMPs) 3 play key roles in these processes (1-3). Mutagenesis has established the importance of the V-B loop (joining ␤-strand V and helix B) in collagenolysis by MMP-1 (4). Residues at the C-terminal end of this loop in MMP-8 were also implicated in collagenolysis (5) and triple helical peptidase activity (6).Binding of an inactivated MMP-1 to an intact collagen fibril likely unwinds the triple helix at the cleavage site to provide access for an active MMP-1 catalytic domain to hydrolyze individual chains (7). The direction of collagen triple helices at the active site is unknown, but the orientation can be hypothesized to be the same as in linear peptide substrates that run antiparallel to ␤-strand IV (8, 9). Single chain peptides from the ␣1 chain of type I/III collagens were soaked into crystals of MMP-12 and MMP-8 catalytic domains, and snapshots of bound hydrolysis products were obtained (8).However, neither crystallography nor NMR data representative of interactions with collagen have been reported because of technical limitations. More generally, structural studies of complexes of enzymes with their substrates have been rare presumably because of catalytic turnover and affinities lower than those for transition state analogues. Several biologically active, self-assembling triple helical peptide (THP) mimics of collagens have been developed. These THPs have facilitated numerous biochemical and cell biological studies otherwise infeasible with insoluble, intact collagen fibrils. THPs are successful in reproducing the cleavage sites of MMPs in collagens I, II, III, V, and XI and in reproducing rate dependences on intact triple helical structure (10, 11). Fluorogenic labeling of THPs has facilitated quantitative comparisons of the triple helical peptidase activities of a variety of MMPs in solution and in cell culture assay (6,11,12). The affinities of MMP catalytic domains for triple helices range from 1 to 80 M (6, 11-13). Collagenolysis involves several MMP functions that include initial nonspecific binding and orientation of collagen fibrils (14, 15), manipulation of collagen fibrils with the C-terminal hemopexin domain (16) and catalytic domain (7), unwinding of triple helices within fibrils, and ensuing hydrolysis of single chains from the melted triple helix (7). THPs are not suitable for modeling the initial binding, orientation, and manipulation of full-length collagen assembled into large, insoluble fibrils (6, 11). They are too small to participate in these higher order events (7). Nonetheless THPs have proven themselves to be outstanding substrates for detailed characterization of the triple helical peptidase activities of MMPs and other metalloprotein...

The Role of Collagen Charge Clusters in the Modulation of Matrix Metalloproteinase Activity

“…The hydrolysis of fTHP-15 by each of these MMPs with the exception of MMP-12 has been reported previously (16 -18, 22, 40) and occurs at the Gly2Leu bond. MMP-12 hydrolysis of fTHP-15 was considerably slower than for other MMPs (Table 2) and much slower than for MMP-12 hydrolysis of an fTHP derived from type V collagen (58). The MMP-12 used herein lacks a C-terminal hemopexin-like domain, which enhances MMP-12 catalytic activity for the type V collagen fTHP (58).…”

“…The MMP-12 used herein lacks a C-terminal hemopexin-like domain, which enhances MMP-12 catalytic activity for the type V collagen fTHP (58). Nonetheless, the MMP-12 catalytic domain possesses collagenolytic activity (57,58) and is the physiologically relevant form of MMP-12 (59), and fTHP-15 serves as a well established point of reference for all of the MMPs.…”

Basis for substrate recognition and distinction by matrix metalloproteinases

“…Second, a SDP subset we identified here in the SV-hB loop (Fig. 4) colocalizes with the region that is required for the collagenolytic activity of several MMPs (35,38,39). This colocalization raises questions as to whether these particular SDPs distinguish the collagenolytic vs. noncollagenolytic MMPs (see below).…”

“…This observation suggested that particular positions contribute to substrate recognition in varying degrees. This idea was tested by calculating the sequence-activity correlation coefficient (SACC), which determines the impact of sequence variability at each position on substrate cleavage (35). An SACC score of 1 indicates the total dominance of substrate recognition by that position, whereas a 0 score indicates a lack of any effect.…”

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

“…We performed molecular modeling of the substrates followed by docking analyses of the catalytic domain of MT1-MMP with the two substrates (details in Materials and Methods). Our docking results were compared with available NMR structures of structurally homologous MMP-collagen systems (24,28), showing that the collagen-like substrates exist in either stretched or bended configurations (SI Appendix, Fig. S1).…”