Inhibition of MMP-2 gelatinolysis by targeting exodomain–substrate interactions

Abstract

MMP-2 (matrix metalloproteinase 2) contains a CBD (collagen-binding domain), which is essential for positioning gelatin substrate molecules relative to the catalytic site for cleavage. Deletion of the CBD or disruption of CBD-mediated gelatin binding inhibits gelatinolysis by MMP-2. To identify CBD-binding sites on type I collagen and collagen peptides with the capacity to compete CBD binding of gelatin and thereby inhibit gelatinolysis by MMP-2, we screened a one-bead one-peptide combinatorial peptide library with recombinant CBD as bait. Analyses of sequences from the CBD-binding peptides pointed to residues 715-721 in human alpha1(I) collagen chain as a binding site for CBD. A peptide (P713) including this collagen segment was synthesized for analyses. In SPR (surface plasmon resonance) assays, the CBD and MMP-2(E404A), a catalytically inactive MMP-2 mutant, both bound immobilized P713 in a concentration-dependent manner, but not a scrambled control peptide. Furthermore, P713 competed gelatin binding by the CBD and MMP-2(E404A). In control assays, neither of the non-collagen binding alkylated CBD or MMP-2 with deletion of CBD (MMP-2DeltaCBD) bound P713. Consistent with the exodomain functions of the CBD, P713 inhibited approximately 90% of the MMP-2 gelatin cleavage, but less than 20% of the MMP-2 activity on a peptide substrate (NFF-1) which does not require the CBD for cleavage. Confirming the specificity of the inhibition, P713 did not alter MMP-2DeltaCBD or MMP-8 activities. These experiments identified a CBD-binding site on type I collagen and demonstrated that a corresponding synthetic peptide can inhibit hydrolysis of type I and IV collagens by competing CBD-mediated gelatin binding to MMP-2.

Molecular mechanism of pancreatic tumor metastasis inhibition by Gd@C 82 (OH) 22 and its implication for de novo design of nanomedicine

“…Phase I: a diffusion-controlled nonspecific electrostatic interaction; phase II: a transient nonspecific hydrophobic interaction; and phase III: a specific hydrophobic and hydrogen-bonded stable binding. (24). The specificity loop has often been focused as a hotspot for selective MMP inhibitors because of its high sequence and length variability in different MMPs.…”

The Collagen Binding Domain of Gelatinase A Modulates Degradation of Collagen IV by Gelatinase B

“…In this regard, it is worth stressing that the reverse investigation (i.e., the synergy of MMP9 CBD and MMP2 in degrading native collagen IV) cannot be studied as the CBD of MMP9 has been reported to bind collagen IV only at high concentrations. [31][32][33][34][35][36] The biological relevance of the reported modulation is validated by the accompanying observations on the effect of this interaction on neutrophil chemokinesis, raising important questions on the strategy for the design of efficient and selective inhibitors against macromolecular substrates.…”

“…27,28 Also, the role of the CBD exosite in modulating type I collagen degradation has been extensively documented. [29][30][31][32][33][34] Nevertheless, despite the importance of type IV collagen degradation, it has been little studied. 35 We have recently shown that MMP2 is able to cleave the α1 and α2 chains of type IV collagen at 37°C and that its CBD is involved in the recognition of both chains by MMP2.…”

Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes

“…That FnII domain binds preferentially to synthetic peptides with a high proportion of aromatic residues and no acidic side chains, and, being a major structural determinant of MMP-2, represents an intriguing pharmacological target (Briknarova et al, 1999;Trexler et al, 2003). The CBD is the main domain involved in substrates recognition (Gioia et al, 2007;Monaco et al, 2006;Patterson et al, 2001); as a matter of fact, MMP-2 mutants lacking the CBD lose their ability to bind to gelatin (Murphy et al, 1994;Xu et al, 2007). Therefore, the CBD-mediated recognition of specific binding sites is essential to drive the correct positioning of the catalytic domain to the adjacent cleavage site.…”