X-ray Absorption Studies of Human Matrix Metalloproteinase-2 (MMP-2) Bound to a Highly Selective Mechanism-based Inhibitor COMPARISON WITH THE LATENT AND ACTIVE FORMS OF THE ENZYME* Received for publication, December 22, 2000 Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M011604200 Oded Kleifeld‡, Lakshmi P. Kotra§¶, David C. Gervasi§, Stephen Brown§¶, M. Margarida Bernardo§, Rafael Fridman§, Shahriar Mobashery§¶, and Irit Sagi‡** From the ‡Department of Structural Biology, The Weizmann Institute of Science, Rehovot 76100, Israel and the §Institute for Drug Design, the ¶Department of Chemistry, Pharmacology and of Biochemistry and Molecular Biology, and the Department of Pathology, Wayne State University, Detroit, Michigan 48202 Malignant tumors express high levels of zinc-depend- ent endopeptidases called matrix metalloproteinases (MMPs), which are thought to facilitate tumor metasta- sis and angiogenesis by hydrolyzing components of the extracellular matrix. Of these enzymes, gelatinases A (MMP-2) and B (MMP-9), have especially been impli- cated in malignant processes, and thus, they have been a target for drugs designed to block their activity. There- fore, understanding their molecular structure is key for a rational approach to inhibitor design. Here, we have conducted x-ray absorption spectroscopy of the full- length human MMP-2 in its latent, active, and inhibited states and report the structural changes at the zinc ion site upon enzyme activation and inhibition. We have also examined the molecular structure of MMP-2 in com- plex with SB-3CT, a recently reported novel mechanism- based synthetic inhibitor that was designed to be highly selective in gelatinases (1). It is shown that SB-3CT di- rectly binds the catalytic zinc ion of MMP-2. Interest- ingly, the novel mode of binding of the inhibitor to the catalytic zinc reconstructs the conformational environ- ment around the active site metal ion back to that of the proenzyme. Zinc-dependent endopeptidases of the family of matrix met- alloproteinases (MMPs) 1 serve important functions in tissue remodeling, organ development, ovulation, fetus implantation, embryogenesis, wound healing, and angiogenesis (2). More- over, various members of the MMP family have been impli- cated in a number of pathological conditions, including cancer growth, tumor angiogenesis, metastasis and arthritis, connec- tive tissue diseases, inflammation, and cardiovascular and au- toimmune diseases (3). Due to the broad spectrum of patholog- ical conditions associated with disregulation of MMP activity, synthetic MMP inhibitors are highly sought (1, 4 – 8). However, the molecular mechanisms of MMP activation and inhibition are still not fully understood (9, 10). X-ray crystal structures are available for the catalytic do- mains of various MMPs (11, 12), including the full-length la- tent MMP-2 (pro-MMP-2) (13). In addition, structures of inhib- itor-enzyme complexes are also available (14 –19). Structural analysis of MMP-inhibitor complexes has mainly focused on the study of the interactions of the catalytic domains of the enzyme with sulfonamide or hydroxamic acid derivatives as zinc-cheating ligands (7, 12, 17, 20). Most of the synthetic inhibitors are designed to provide a bidentate chelating ligand to the catalytic zinc ion. It is a general trend that these metal chelators largely lack selectivity in inhibition of MMPs. This complicates the possibility for targeting specific members of the MMP family in a particular pathological condition. A recent report by Brown et al. (1) described a novel concept for the selective inhibition of gelatinases (MMP-2 and MMP-9) by the design and synthesis of the first mechanism-based MMP inhib- itor (“suicide substrate”) for any MMP. This small molecule inhibitor, designated SB-3CT, provides a potent and highly selective inhibition of human gelatinases by the manifestation of both slow binding and mechanism-based inhibition behavior in its kinetic profile (1). To gain insight into the mechanism of inhibition of MMP-2 by SB-3CT and the local structure around the catalytic zinc ion in latent, active, and inhibited MMP-2, the zinc ion coordina- tion shell in all complexes was studied by x-ray absorption spectroscopy (XAS). Our results show that the catalytic zinc ion is directly coordinated to the sulfur atom of the bound inhibitor in a monodentate manner to form a tetrahedral coordination at the zinc ion. Interestingly, the inhibited enzyme retains the conformation of the latent MMP-2 around the zinc coordination shell, which may explain the remarkable selectivity of SB-3CT for gelatinases for which it was designed. EXPERIMENTAL PROCEDURES Human pro-MMP-2 was expressed in a recombinant vaccinia virus mammalian cell expression system, as described earlier (21). Pro- MMP-2 was purified to homogeneity from the media of infected HeLa cells by gelatin-agarose affinity chromatography, as described previ- ously (21). The protein concentration of proMMP-2 was determined using the molar extinction coefficient of 122,800 M -1 cm -1 (22) and amino acid analysis. Pro-MMP-2 was activated by 1 mM p-aminophe- nylmercuric acetate (dissolved in 200 mM Tris) for 30 min at 37 °C. The protein concentration of the active MMP-2 was determined by titration with TIMP-2, as described previously (23). The synthetic mechanism-based inhibitor was synthesized for these studies as described earlier (1). MMP-2 was inhibited for our studies according to the procedure that has been reported previously (1). * This work was supported by Grants 6602/2 from the Binational Scientific Foundation and the Maurizio and Clotilde Pontecorvo Fund (to I. S.), Grant DAMD17-97-1-7174 from the United States Army (to S. M.), and Grants CA-61986 and CA-82298 from the National Insti- tutes of Health (to R. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ** To whom correspondence should be addressed: Dept. of Structural Biology, The Weizmann Institute of Science, Rehovot 76100, Israel. Tel.:/Fax: 972-8-934-2130; E-mail: irit.sagi@weizmann.ac.il. 1 The abbreviations used are: MMP, matrix metalloproteinase; XAS, x-ray absorption spectroscopy; ICP-AES, inductively coupled plasma atomic emission spectroscopy; XANES, x-ray absorption near edge structure; EXAFS, extended x-ray absorption fine structure; TIMP, tissue inhibitor metalloproteinase. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 20, Issue of May 18, pp. 17125–17131, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. This paper is available on line at http://www.jbc.org 17125 by guest on June 30, 2016 http://www.jbc.org/ Downloaded from