Basis for substrate recognition and distinction by matrix metalloproteinases

Abstract

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...

Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

“…Such methods include engineering deletion mutants (3), use of competitive ligands (4,5), and site-directed mutagenesis (6,7). In contrast to these techniques, substrate phage display is a highthroughput, unbiased approach to studying protease substrate specificity (8)(9)(10). In this method, a library consisting of 10 6 -10 9 independent phage clones, each expressing a unique potential substrate on its surface, is panned for multiple rounds with a protease, and the cleaved or uncleaved phages after each reaction are removed and amplified for subsequent rounds of selection.…”

Matrix Metalloproteinase (MMP) Proteolysis of the Extracellular Loop of Voltage-gated Sodium Channels and Potential Alterations in Pain Signaling

“…Recently, using substrate phage display (17,18), we identified some of the cleavage preferences of the matrix metalloproteinase (MMP) family members (19). Here, we employed a high throughput multiplexed peptide-centric profiling technology involving the cleavage of 18,583 peptides by the 18 individual proteinases from the main subgroups of the MMP family (20).…”

“…The algorithm is based on the position weight matrices (PWMs) approach. The original cleavage data were derived from the analysis of specific substrates selected from phage display libraries (17,18).…”

rs3918242 MMP9 gene polymorphism is associated with myocardial infarction in Mexican patients

“…The MMPs are a family of zinc-dependent endopeptidases responsible for degrading extracellular components (Creemers et al, 2001;Falk, 2006). Each MMP has its own substrate specificity (Ratnikov et al, 2014). Previous studies have suggested that MMPs may play a role in the development of several diseases, such as spontaneous early pregnancy failure (Nissi et al, 2013), solid and hematological malignancies (Chaudhary et al, 2013), and abdominal aortic aneurysms (Duellman et al, 2012).…”