Abstract
Unlike other synthetic or physiological inhibitors for matrix metalloproteinases (MMPs), the β-amyloid precursor protein-derived inhibitory peptide (APP-IP) having an ISYGNDALMP sequence has a high selectivity toward MMP-2. Our previous study identified amino acid residues of MMP-2 essential for its selective inhibition by APP-IP and demonstrated that the N to C direction of the decapeptide inhibitor relative to the substrate-binding cleft of MMP-2 is opposite that of substrate. However, detailed interactions between the two molecules remained to be clarified. Here, we determined the crystal structure of the catalytic domain of MMP-2 in complex with APP-IP. We found that APP-IP in the complex is indeed embedded into the substrate-binding cleft of the catalytic domain in the N to C direction opposite that of substrate. With the crystal structure, it was first clarified that the aromatic side chain of Tyr(3) of the inhibitor is accommodated into the S1' pocket of the protease, and the carboxylate group of Asp(6) of APP-IP coordinates bidentately to the catalytic zinc of the enzyme. The Ala(7) to Pro(10) and Tyr(3) to Ile(1) strands of the inhibitor extend into the nonprime and the prime sides of the cleft, respectively. Therefore, the decapeptide inhibitor has long range contact with the substrate-binding cleft of the protease. This mode of interaction is probably essential for the high MMP-2 selectivity of the inhibitor because MMPs share a common architecture in the vicinity of the catalytic center, but whole structures of their substrate-binding clefts have sufficient variety for the inhibitor to distinguish MMP-2 from other MMPs.
Highlights
The matrix metalloproteinases (MMPs)2 comprise a family of zinc-dependent endopeptidases capable of degrading protein components of the extracellular matrix and play pivotal roles in tissue remodeling under physiological and pathological conditions such as morphogenesis, angiogenesis, tissue repair, and tumor invasion [1,2,3,4]
To clarify the mechanism of the selective inhibition, we have determined the amino acid residues of MMP-2 essential for its interaction with Amyloid precursor protein (APP)-IP by analyzing APPderived inhibitory peptide (APP-IP) inhibitions of various chimeric mutants of MMPs, and we have revealed that several residues of MMP-2 located far from the catalytic zinc in the nonprime or the prime side of the substrate-binding cleft are essential for the selective interaction [24]
Primary Structure and Numbering for Amino Acid Residues of Mutant Form of the Catalytic Domain of MMP-2 Co-crystalized with APP-IP—We first tried to co-crystalize the catalytic domain of MMP-2 with APP-IP by extensive screening, but no crystal was obtained
Summary
Materials—The sources of materials used are as follows: pFLAG-CTC vector from Sigma. cDNA of human proMMP-2 was cloned into pCMV6 vector from OriGene Technologies (Rockville, MD). To remove the cording region of the three fibronectin type II domains, PCR with PrimeStar Max DNA polymerase was carried out, using a pair of primers 5Ј-GGTACAGCCTGTTCCTCG-3Ј (sense primer) and 5Ј-CTTGGCCTTCTCCCAAGG-3Ј (antisense primer) and the pFLAG-N-ins-MMP-2-cat-FN as a template. To introduce a mutation that substitutes Glu121, the active site residue of MMP-2, with Ala, PCR with PrimeStar Max DNA polymerase was carried out, using a pair of primers 5Ј-GCCCACGCGTTTGGCCACGCCATGG-3Ј (sense primer) and 5Ј-GCCAAACGCGTGGGCTGCCACGAGG-3Ј (antisense primer) and the pFLAG-N-insMMP-2-cat as a template. To introduce mutations in a loop region of the catalytic domain of MMP-2, by which crystallization of the recombinant protein is probably facilitated, PCR with PrimeStar Max DNA polymerase was carried out, using a pair of primers 5Ј-GGAAAAGGCGTTGGGTACAGCCTGTTCCTC3Ј (sense primer) and 5Ј-CCCAACGCCTTTTCCCAAGGTCCATAGCTCA-3Ј (antisense primer) and the pFLAG-N-insMMP-2-cat (E121A) as a template. Prior to x-ray experiments, crystals were transferred to cryoprotectant consisting
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