NMR and Bioinformatics Discovery of Exosites That Tune Metalloelastase Specificity for Solubilized Elastin and Collagen Triple Helices

Abstract

The catalytic domain of metalloelastase (matrix metalloproteinase-12 or MMP-12) is unique among MMPs in exerting high proteolytic activity upon fibrils that resist hydrolysis, especially elastin from lungs afflicted with chronic obstructive pulmonary disease or arteries with aneurysms. How does the MMP-12 catalytic domain achieve this specificity? NMR interface mapping suggests that ␣-elastin species cover the primed subsites, a strip across the ␤-sheet from ␤-strand IV to the II-III loop, and a broad bowl from helix A to helix C. The many contacts may account for the comparatively high affinity, as well as embedding of MMP-12 in damaged elastin fibrils in vivo. We developed a strategy called BINDSIght, for bioinformatics and NMR discovery of specificity of interactions, to evaluate MMP-12 specificity without a structure of a complex. BINDSIght integration of the interface mapping with other ambiguous information from sequences guided choice mutations in binding regions nearer the active site. Single substitutions at each of ten locations impair specific activity toward solubilized elastin. Five of them impair release of peptides from intact elastin fibrils. Eight lesions also impair specific activity toward triple helices from collagen IV or V. Eight sites map to the "primed" side in the III-IV, V-B, and S1 specificity loops. Two map to the "unprimed" side in the IV-V and B-C loops. The ten key residues circumscribe the catalytic cleft, form an exosite, and are distinctive features available for targeting by new diagnostics or therapeutics.Although protein-protein interactions are universally important, mechanistic understanding of their specificity is often poor (1). An impediment to detailed understanding of proteolytic attack of proteins is the transience and potential heterogeneity of the interactions, which interfere in capturing the structure of a substrate complex by crystallography or other methods. These complications affect characterization of matrix metalloproteinase-12 (MMP-12), 3 the metalloelastase secreted by human macrophages at sites of inflammation. To investigate how MMP-12 achieves specificity for protein fibrils from lungs and arteries, we developed an approach designated BINDSIght, for its combination of bioinformatics and NMR discovery of specificity of interactions.In lungs, arteries, skin, and basement membranes, elastin provides elastic recoil, is heavily cross-linked, and is difficult to digest. Collagens are ubiquitous and comprise ϳ25% of the protein mass of the body. Damage to fibrils of the extracellular matrix by proteases such as MMP-12 contributes to the inflammation and chronic disease states of chronic obstructive pulmonary disease (2-4), atherosclerosis (5-6), abdominal aortic aneurysm (7), multiple sclerosis (8), ulcerative colitis (9), asthma (4), and rheumatoid arthritis (10). The progression of chronic obstructive pulmonary disease/emphysema and (abdominal aortic aneurysm) in smokers depends in large part on MMP-12 expression (11) and its degradation of elastin (7, 12). ...

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

“…Apart from the Hpx domains of collagenases, examples include the fibronectin type II repeats in MMP-2 and MMP-9, which contribute to the binding and degradation of elastin, gelatin, and type IV collagen (47). Recent NMR and mutagenesis studies of the Cat domain of MMP-12 characterized several exosites for elastin (48,49). Similar studies were carried out with the isolated Hpx domain of MMP-1 and a triple-helical collagen peptide (40) and suggested a role for Phe282 (Phe301 in the numbering used in ref.…”

Exosite Interactions Impact Matrix Metalloproteinase Collagen Specificities

“…MMP-1 residue 210 (and the corresponding residue in MMP-8) facilitates collagenolytic and triple-helical peptidase activities (8,46). When studying macrophage elastase (MMP-12) interactions with triple-helical substrates by NMR spectroscopy, several CAT domain exosites were identified and localized (47,48). For example, analyzing MMP-12 alone or in complex with the ␣1(V)436 -450 THP showed interactions located far from the catalytic cleft; more than 30 residues total were altered from the uncomplexed enzyme, especially Asp-124, Asp-164, and .…”

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

“…However, MMP-12 cannot have the same extended interactions with substrate as MMP-2 and MMP-9 because MMP-12 does not possess the fibronectin type II inserts that MMP-2 and MMP-9 utilize to bind collagens (1). Prior studies showed that the MMP-12 catalytic domain possesses collagenolytic activity (57), and this domain provides contacts outside of the active site cleft (exosites) that participate in collagenolysis (58,74). As one of the proposed contacts involves Lys 241 of MMP-12, its interactions with charge clusters might differ from those of the collagenases (MMP-1, MMP-8, MMP-13, and MT1-MMP) and gelatinases (MMP-2 and MMP-9).…”