Differentiation of Secreted and Membrane-Type Matrix Metalloproteinase Activities Based on Substitutions and Interruptions of Triple-Helical Sequences

Minond, Dmitriy; Lauer‐Fields, Janelle L.; Čudić, Mare; Overall, Christopher M.; Pei, Duanqing; Brew, Keith; Moss, Marcia L.; Fields, Gregg B.

doi:10.1021/bi062199j

Cited by 47 publications

(64 citation statements)

References 74 publications

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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: 97%

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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Section: Discussionmentioning

confidence: 97%

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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“…However, the interaction of collagen with MMPs extends over a significant length of the triple helix as modulation of MMP-1, MMP-8, MMP-13, and MT1-MMP activity was observed in THP substrates spanning subsites P 13 -P 17 Ј (17,18,22,26). X-ray crystallographic and NMR spectroscopic studies of MMP-1⅐THP complexes also revealed extended interactions between enzyme and substrate (28,29).…”

mentioning

confidence: 99%

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

Lauer

Bhowmick

Tokmina‐Roszyk

et al. 2014

Journal of Biological Chemistry

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Background:The role of charged clusters in modulating MMP hydrolysis of collagen is unknown. Results: Triple-helical peptides containing charged motifs were as good or better substrates than the parent (non-chargeclustered) peptide. Conclusion: MMP-2 and MMP-9 greatly favored the presence of charged residues. Significance: The lack of Gly-Asp-Lys clusters in native collagens may diminish potential MMP-2 and MMP-9 collagenolytic activity.

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“…Typically, the P 5 ′ position of the fTHP accommodates Lys(Dnp) while the P 5 position accommodates Lys(Mca) (seenote 1). fTHPs have subsequently provided insights into (a) the relative triple-helical peptidase activities of the various collagenolytic MMPs, (b) the collagen preferences of these MMPs, and (c) the relative roles of MMP domains and specific residues in efficient collagenolysis (50,52,53,57,58,(60)(61)(62)(63)(64)(65)(66). fTHPs have been noted as being particularly advantageous for dissecting the mechanism of collagenolysis (8).…”

Section: Triple-helicalmentioning

confidence: 99%

“…The fTHP substrates have been utilized to determine individual kinetic parameters (Table 1) and activation energies for hydrolysis of triple-helices (57,58,61,(63)(64)(65). MMP-1, MMP-2, MMP-8, MMP-9, MMP-13 and MMP-14 hydrolysis of the consensus types I-III collagen sequence (designated fTHP-3 and fTHP-4) occurred at the Gly~Leu bond, the analogous bond cleaved by these MMPs in the native collagen chains (6).…”

Section: Triple-helicalmentioning

confidence: 99%

“…As noted by George et al for HTS of MMP-3, the CyDye pair of Cy3/Cy5Q [for the substrate acetyl-Arg-Pro-Lys(Cy3)-Pro-Val-Glu~Nva-Trp-Arg-Lys(Cy5Q)-NH 2 ] was much less susceptible to false results than the Mca/Dnp pair, as <1% of a random library were auto-fluorescent at Cy3 wavelengths while >10% of the same library could not be screened using Mca/Dnp due to auto-fluorescence and interference (36). fTHP assays have been utilized with 96-, 384-, and 1536-well plates (57,58,(60)(61)(62)(63)(64)(65)(81)(82)(83). Because the THPs have distinct conformational features that interact with protease secondary binding sites (exosites) (82), these substrates can be utilized for the identification of non-active site binding inhibitors.…”

Section: Fluorogenic Substrates For High-throughput Screening (Hts)-thementioning

confidence: 99%

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Using Fluorogenic Peptide Substrates to Assay Matrix Metalloproteinases

Fields

Matrix Metalloproteinase Protocols

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A continuous assay method, such as one that utilizes an increase in fluorescence upon hydrolysis, allows for rapid and convenient kinetic evaluation of proteases. To better understand MMP behaviors and to aid in the design of MMP inhibitors, a variety of sequence specificity, phage display, and combinatorial chemistry studies have been performed. Results of these studies have been valuable for defining the differences in MMPs and for creating quenched fluorescent substrates that utilize fluorescence resonance energy transfer (FRET)/intramolecular fluorescence energy transfer (IFET). FRET triple-helical substrates have been constructed to examine the collagenolytic activity of MMP family members. The present chapter provides an overview of MMP and related FRET substrates and describes how to construct and utilize these substrates.

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Differentiation of Secreted and Membrane-Type Matrix Metalloproteinase Activities Based on Substitutions and Interruptions of Triple-Helical Sequences

Cited by 47 publications

References 74 publications

Basis for substrate recognition and distinction by matrix metalloproteinases

Basis for substrate recognition and distinction by matrix metalloproteinases

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

Using Fluorogenic Peptide Substrates to Assay Matrix Metalloproteinases

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