The Roles of Substrate Thermal Stability and P2 and P1′ Subsite Identity on Matrix Metalloproteinase Triple-helical Peptidase Activity and Collagen Specificity

Minond, Dmitriy; Lauer‐Fields, Janelle L.; Čudić, Mare; Overall, Christopher M.; Pei, Duanqing; Brew, Keith; Visse, Robert; Nagase, Hideaki; Fields, Gregg B.

doi:10.1074/jbc.m606004200

Cited by 89 publications

(144 citation statements)

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“…Elastins and collagens should spill out of the cleft. Consistent with this, the triple helical peptidase activity of MMP-1, -8, and -12 involves the V-B loop preceding the active-site helix B (33,(45)(46)(47). A triple helical peptide substrate derived from collagen V contacts not only this exosite but also appears to cover other sites in loops and atop the ␤-sheet even more remote from the active site cleft of MMP-12 (33).…”

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confidence: 67%

“…The lesions' impact on specific activities for substrates from type IV and V collagen raises an important question: Do the corresponding positions in orthodox collagenolytic MMPs modulate their turnover of collagen triple helices? This possibility is suggested by MMP-1 and -8 relying on the V-B loop (45)(46)(47). The exosite of Fig.…”

Section: Of Ref 33)mentioning

confidence: 99%

“…Yet MMP activity upon protein substrates is a much different and more physiological context for considering specificity. For example, the V-B loop influences triple helical peptidase activity of MMP-1, -8, and -12 (33,(45)(46)(47). About 30 of the distinctive sequence positions might contact 20-kDa ␣-elastin species (Eln-20) in (Fig.…”

Section: Mmp-12 Specificity For Substrates From Fibrils Engagesmentioning

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

Journal of Biological Chemistry

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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). ...

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confidence: 67%

Section: Of Ref 33)mentioning

confidence: 99%

Section: Mmp-12 Specificity For Substrates From Fibrils Engagesmentioning

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

Journal of Biological Chemistry

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“…In addition to the impact of ancillary domains in binding collagen, cleavage of this complex substrate is fundamentally different from hydrolysis of linear peptide/protein substrates by MMPs. This distinction is illustrated by work with triple helical peptides that model collagen structure (45)(46)(47). It will be interesting to know how the SDPs identified here contribute to this multistep process of collagen cleavage, which is unique to only a few MMPs.…”

Section: Discussionmentioning

confidence: 99%

Basis for substrate recognition and distinction by matrix metalloproteinases

Ratnikov¹,

Cieplak²,

Gramatikoff³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Genomic sequencing and structural genomics produced a vast amount of sequence and structural data, creating an opportunity for structure-function analysis in silico [Radivojac P, et al. (2013) Nat Methods 10(3):221-227]. Unfortunately, only a few large experimental datasets exist to serve as benchmarks for functionrelated predictions. Furthermore, currently there are no reliable means to predict the extent of functional similarity among proteins. Here, we quantify structure-function relationships among three phylogenetic branches of the matrix metalloproteinase (MMP) family by comparing their cleavage efficiencies toward an extended set of phage peptide substrates that were selected from ∼64 million peptide sequences (i.e., a large unbiased representation of substrate space). The observed second-order rate constants [k (obs) ] across the substrate space provide a distance measure of functional similarity among the MMPs. These functional distances directly correlate with MMP phylogenetic distance. There is also a remarkable and near-perfect correlation between the MMP substrate preference and sequence identity of 50-57 discontinuous residues surrounding the catalytic groove. We conclude that these residues represent the specificity-determining positions (SDPs) that allowed for the expansion of MMP proteolytic function during evolution. A transmutation of only a few selected SDPs proximal to the bound substrate peptide, and contributing the most to selectivity among the MMPs, is sufficient to enact a global change in the substrate preference of one MMP to that of another, indicating the potential for the rational and focused redesign of cleavage specificity in MMPs.protease | specificity-determining positions | MMPs A paramount objective of biological research is to understand how sequence encodes function. Previously, functional regions in proteins were identified using large-scale mutagenesis (e.g., alanine scanning) (1). More recently, our insights are largely gained by computational approaches aimed at comparing sequences and structures of large protein sets across multiple genomes. The vast increase in the number of available sequences makes it possible to compare homology between sequences from genome projects to proteins of known structure and function and, as a result, identify functional similarities in silico (2-4). Because the global fold of most ordered proteins can be reliably predicted (5, 6) and because the catalytic residues of most classes of enzymes are either known or can be inferred (7,8), protein sequences can now be directly used to elucidate and classify major protein functions (9-12), and are even being extended to predict enzyme substrates (13).However, such classifications fail to explain the specialization and expansion of function that is required for organismal plasticity, complexity, and adaptability, all of which are normally driven by gene duplications and subsequent divergence. Computational approaches aimed at identifying functional distinctions across protein families have pri...

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“…However, several members of the matrix metalloproteinase (MMP) 2 subfamily of zinc-dependent endopeptidases catalyze the hydrolysis of triple helices (5). Although highly homologous, MMPs have distinct preferences among collagenous substrates (5,8). MMP-1, MMP-8, MMP-13, and MMP-14/ membrane-type 1 MMP (MT1-MMP) are collagenases that hydrolyze the triple helix of fibrillar type I-III collagens with varying efficiencies at a single GlyϳIle/Leu site.…”

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confidence: 99%

Exosite Interactions Impact Matrix Metalloproteinase Collagen Specificities

Robichaud

Steffensen

Fields

2011

Journal of Biological Chemistry

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Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. However, the substrate structural determinants that facilitate interaction with specific MMPs are not well defined. We hypothesized that type I-III collagen sequences located N-or C-terminal to the physiological cleavage site mediate substrate selectivity among MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14/membrane-type 1 (MT1)-MMP. The enzyme kinetics for hydrolysis of three fluorogenic triple-helical peptides (fTHPs) was evaluated herein. The first fTHP contained consensus residues 769 -783 from type I-III collagens, the second inserted ␣1(II) collagen residues 763-768 N-terminal to the consensus sequence, and the third inserted ␣1(II) collagen residues 784 -792 C-terminal to the consensus sequence. Our analyses showed that insertion of the C-terminal residues significantly increased k cat /K m and k cat for MMP-1. MMP-13 showed the opposite behavior with a decreased k cat /K m and k cat and a greatly improved K m in response to the C-terminal residues. Insertion of the N-terminal residues enhanced k cat /K m and k cat for MMP-8 and MT1-MMP. For MMP-2, the C-terminal residues enhanced K m and dramatically decreased k cat , resulting in a decrease in the overall activity. These changes in activities and kinetic parameters represented the collagen preferences of MMP-8, MMP-13, and MT1-MMP well. Thus, interactions with secondary binding sites (exosites) helped direct the specificity of these enzymes. However, MMP-1 collagen preferences were not recapitulated by the fTHP studies. The preference of MMP-1 for type III collagen appears to be primarily based on the flexibility of the hydrolysis site of type III collagen compared with types I and II. Further characterization of exosite determinants that govern interactions of MMPs with collagenous substrates should aid the development of pharmacotherapeutics that target individual MMPs.

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The Roles of Substrate Thermal Stability and P2 and P1′ Subsite Identity on Matrix Metalloproteinase Triple-helical Peptidase Activity and Collagen Specificity

Cited by 89 publications

References 91 publications

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

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

Basis for substrate recognition and distinction by matrix metalloproteinases

Exosite Interactions Impact Matrix Metalloproteinase Collagen Specificities

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