Basis for substrate recognition and distinction by matrix metalloproteinases

Significance Specificity-determining positions (SDPs) account for distinctions in function across a protein family. Many theories on the evolution of functional specificity have led to approaches for predicting SDPs in silico, but large experimental datasets allowing a statistical assignment are lacking. Here, the SDPs of matrix metalloproteinases are elucidated by querying the proteolytic efficiency of eight matrix metalloproteinases, representing three phylogenetic branches, in an extended and diverse substrate space. More than 10,000 measures of cleavage efficiency reveal a near-perfect correlation between similarity in proteolytic function and sequence identity at 50–57 positions on the front face of the catalytic domain. These positions are assigned as SDPs. Transmutation of proteolytic function is possible by swapping SDPs nearest to bound substrate. 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 function-related 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.