The 1.8-angstrom crystal structure of a matrix metallaproteinase 8-barbiturate inhibitor complex reveals a previously unobserved mechanism for collagenase substrate recognition

The individual zinc endoproteinases of the tissue degrading matrix metallop roteinase (MMP) family share a common catalytic architecture but are differ entiated with respect to substrate specificity, localization, and activatio n. Variation in domain structure and more subtle structural differences con trol their characteristic specificity profiles for substrates from among fo ur distinct classes (Nagase, H., and Woessner, J. F. J. (1999) J. Biol. Che m. 274, 21491-21494). Exploitation of these differences may be decisive for the design of anticancer or other drugs, which should be highly selective for their particular MMP targets. Based on the 1.8-Angstrom crystal structu re of human neutrophil collagenase (MMP-8) in complex with an active site-d irected inhibitor (RO200-1770), we identify and describe new structural det erminants for substrate and inhibitor recognition in addition to the primar y substrate recognition sites. RO200-1770 induces a major rearrangement at a position relevant to substrate recognition near the MMP-8 active site (Al a(206)-Asn(218)). In stromelysin (MMP-3), competing stabilizing interaction s at the analogous segment hinder a similar rearrangement, consistent with kinetic profiling of several MMPs. Despite the apparent dissimilarity of th e inhibitors, the central 2-hydroxypyrimidine-4,6-dione (barbiturate) ring of the inhibitor RO200-1770 mimics the interactions of the hydroxamate-deri ved inhibitor batimastat (Grams, F., Reinemer, P., Powers, J. C., Kleine, T ., Pieper, RI., Tschesche, H., Huber, R., and Bode, W. (1995) fur. J. Bioch em. 228, 830-841) for binding to MMP-8. The two additional phenyl and piper idyl ring substituents of the inhibitor bind into the S1 ' and S2 ' pockets of MMP-8, respectively. The crystal lattice contains a hydrogen bond betwe en the O-gamma group of Ser(209) and N-delta1 of His(207) of a symmetry rel ated molecule; this interaction suggests a model for recognition of hydroxy prolines present in physiological substrates. We also identify a collagenas e-characteristic cis-peptide bond, Asn(188)-Tyr(189), on a loop essential f or collagenolytic activity. The sequence conservation pattern at this posit ion marks this cis-peptide bond as a determinant for triple-helical collage n recognition and processing.