TIMP-1 CONTACT SITES AND PERTURBATIONS OF STROMELYSIN-1 MAPPED BY NMRAND A PARAMAGNETIC SURFACE PROBE

Surfaces of the 173 residue catalytic domain of human matrix metallopr oteinase 3 (MMP-3(Delta C)) affected by binding of the N-terminal, 126 residue inhibitory domain of human TIMP-1 (N-TIMP-1) have been invest igated using an amide-directed, NMR-based approach. The interface was mapped by a novel method that compares amide proton line broadening by paramagnetic Gd-EDTA in the presence and absence of the binding partn er. The results are consistent with the X-ray model of the complex of MMP-3(Delta C) with TIMP-1 (Gomis-Ruth et al. (1997) Nature 389, 77-81 ). Residues Tyr155, Asn162, Val163, Leu164, His166, Ala167, Ala169, an d Phe210 of MMP-3(Delta C) ate protected from broadening by the Gd-EDT A probe by binding to N-TIMP-1. N-TIMP-1-induced exposure of backbone amides of Asp238, Asn240, Gly241, and Ser244 of helix C of MMP-3(Delta C) to Gd-EDTA confirms that the displacement of the N-terminus of MMP -3(Delta C) occurs not only in the crystal but also in solution. These results validate comparative paramagnetic surface probing as a means of mapping protein-protein interfaces. Novel N-TIMP-1-dependent change s in hydrogen bonding near the active site of MMP-3(Delta C) are repor ted. N-TIMP-1 binding causes the amide of Tyr223 of MMP-3(Delta C;) bo und by N-TIMP-1 to exchange with water rapidly, implying a lack of the hydrogen bond observed in the crystal structure. The backbone amide p roton of Asn162 becomes protected from rapid exchange upon forming a c omplex with N-TIMP-1 and could form a hydrogen bond to N-TIMP-1. N-TIM P-1 binding dramatically increases the rate of amide hydrogen exchange of Asp177 of the fifth beta strand of MMP-3(Delta C), disrupting its otherwise stable hydrogen bond.