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 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.

Keywords: MMPs; protease; specificity-determining positions.

Fig. 1.

MMP substrate recognition profiles correlate with phylogenetic distances between catalytic domains. (A) Phylogeny of the catalytic domains of mammalian MMPs. Amino acid sequences of 600 MMPs from different species were aligned using JalView, and a near joint tree was generated using HyperTree algorithm for the catalytic domains (see Materials and Methods for details). Representative members from each of the three major branches were selected for the study: membrane type I MMPs (transmembrane) MMP-14, -15, -16, and -24; gelatinases MMP-2 and -9; and membrane GPI-anchored MMP-17 and -25. (B) Average linkage trees of sequence identity among the catalytic domains of the MMPs (blue) and k_(obs) values obtained for each MMP using the 1,369 phage substrates (orange) were generated using JalView and hierarchical clustering editor (HCE) programs as described in Materials and Methods. The heat map displays the k_(obs) of each MMP for each phage peptide substrate.

Fig. 2.

Distinctions in substrate recognition profiles follow MMP phylogeny. (A) Sequence logos (37) were generated for phage substrates cleaved equally well by the entire set of MMPs (Common) and those selective for individual subfamilies (MT-MMPs, gelatinases, and GPI-anchored MMPs). The height and vertical position of each letter is proportional to the frequency of occurrence of amino acid residues at each position of substrates from P5 to P2′. (B) The contribution of individual subsites to substrate recognition by the test MMPs was determined based on sequence–activity correlation coefficient (SACC) (see SI Materials and Methods for equations) values calculated using k_(obs) data obtained for each MMP against 1,369 phage substrates. SACC ranges from 0 (low) to 1 (high). (C) Heat maps of residue preference (RP) (described in SI Materials and Methods) show how individual residues at specific positions from P5 to P2′ influence k_(obs). The heat map was generated by hierarchical clustering. RP ranges from 0 (low) to 2 (high).

Fig. 3.

Sequence phylogeny of the variable regions of the front face of the catalytic cleft of MMPs directly correlates with functional phylogeny. The degree of sequence identity vs. HR—the pairwise hydrolysis correlation coefficient [defined as Pearson correlation coefficients of k_(obs) values]—was plotted for each pair of MMPs using the sequence of the whole catalytic domain (A), 50–57 variable residues on the binding face of the catalytic domain (red) (B), and the entire catalytic domain exclusive of the areas identified in B (blue) (C). Equations describing individual correlations are shown above each plot. The green spheres in structures represent calcium ions, and the gray ones, zinc ions.

Fig. 4.

SDPs of the MMP catalytic domains are located in the variable surface loops. (A) Sequences of catalytic domains of human MMPs were aligned using JalView and variable residues in the surface loop regions surrounding the catalytic cleft were identified using sequence variability at each position, along with existing structural information or structural models as described in Materials and Methods. The colored regions correspond to the residues comprising the SDPs. Location of the residues relative to the catalytic cleft surface loops is indicated by brackets below the alignment. (B) Localization of the SDPs in 3D space is marked in red on a ribbon diagram of MMP-2.

Fig. 5.

Swapping dominant SDPs transmutes selectivity of MMP-16 into that of MMP-17. (A) Structures of the catalytic domain of MMP-16 (Left) and MMP-17 (Right) with optimal peptide substrates (acetyl-Leu-Val-Pro-Arg-His↓Leu-Phe-Ala-Ser-Gly-N-methyl and acetyl-Arg-Val-Val-Met-Arg↓Leu-Val-Leu-Ser-Gly-N-methyl, respectively) docked into the active site groove as described in Materials and Methods. Regions of the catalytic domains that are SDPs are colored light blue or purple if they were chosen for mutation. The substrate side chains are colored to indicate the SACC score for the corresponding positions in the catalytic cleft (S3–S1). So residues in darker red are at substrate positions with high SACC scores (Fig. 2B), and lighter red indicates residues at positions with lower SACC scores. The remainder of the docked substrate (outside of S3–S1) is colored dark gray. (B) The effect of the transmutation on substrate selectivity was determined using ∼140 phage substrates about one-half of which were selective for each protease. The k_(obs) for each substrate was measured as described in Materials and Methods. Error bars represent SEMs. (C) Sequence logos indicating the frequency of residues at each position calculated for substrates selective for MMP-16 (Left) and those preferentially cleaved by the 16/17 transmutant and MMP-17 (Right).