Solution Structure of Inhibitor-Free Human Metalloelastase (MMP-12) Indicates an Internal Conformational Adjustment

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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). ...

“…Use of E219A-inactivated MMP-12 prevented digestion of Eln-20. The extent of this structural perturbation is localized to residue 219 in the active site (49,(71)(72).…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

Palmier

Fulcher

Bhaskaran

et al. 2010

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“…However, even by using a molecular model describing the potential binding mode of 1 within the hMMP-12 active site, it would be hard to predict the results reported in this study. The main reason for this is the fact that Lys 241 is located on a loop segment of hMMP-12, which, based on both x-ray and NMR studies of free hMMP-12 or in complex with synthetic inhibitors, displays high flexibility with the lysine side chain exhibiting high mobility (33)(34)(35). Superimposition of two hMMP-12 structure models, taken from an ensemble of twenty NMR-derived free hMMP-12 structures (34), provides some clues about the conformational space sampled by the Lys 241 side chain (Fig.…”

Section: Discussionmentioning

confidence: 99%

Molecular Determinants of Matrix Metalloproteinase-12 Covalent Modification by a Photoaffinity Probe

Dabert-Gay

Czarny

Devel

et al. 2008

Mass spectroscopy, microsequencing, and site-directed mutagenesis studies have been performed to identify in human matrix metalloelastase (hMMP-12) residues covalently modified by a photoaffinity probe, previously shown to be able to covalently label specifically the active site of matrix metalloproteinases (MMPs). Results obtained led us to conclude that photoactivation of this probe in complex with hMMP-12 affects a single residue in human MMP-12, Lys 241 , through covalent modification of its side chain ⑀ NH 2 group. Because x-ray and NMR studies of hMMP-12 indicate that Lys 241 side chain is highly flexible, our data reveal the existence of particular Lys 241 side-chain conformation in which the ⑀ NH 2 group points toward the photolabile group of the probe, an event explaining the high levels of cross-linking yield between hMMP-12 and the probe. Lys 241 is not conserved in MMPs, thus differences in cross-linking yields observed with this probe between MMP members may be linked to the residue variability observed at position 241 in this family.

“…Attempts at crystallizing an MMP with THP bound may have been stymied by moderate affinities of MMPs for THPs and complexities of the binding modes of the THPs. The solution structure of MMP-12 with the active site vacant but inactivated by the E219A mutation was recently determined for supporting studies of substrate interactions (36). The moderate K m values of MMPs for THPs suggest that NMR can be a worthwhile avenue to interaction studies because NMR methods accommodate moderate affinities and transient associations (37).…”

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confidence: 99%

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

Bhaskaran

Palmier

Lauer‐Fields

et al. 2008

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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...