title: TIMP-1 contact sites and perturbations of stromelysin 1 mapped by NMR and a paramagnetic surface probe.
abstract: Surfaces of the 173 residue catalytic domain of human matrix metalloproteinase 3 (MMP-3(DeltaC)) affected by binding of the N-terminal, 126 residue inhibitory domain of human TIMP-1 (N-TIMP-1) have been investigated 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 partner. The results are consistent with the X-ray model of the complex of MMP-3(DeltaC) with TIMP-1 (Gomis-Rüth et al. (1997) Nature 389, 77-81). Residues Tyr155, Asn162, Val163, Leu164, His166, Ala167, Ala169, and Phe210 of MMP-3(DeltaC) are protected from broadening by the Gd-EDTA 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(DeltaC) to Gd-EDTA confirms that the displacement of the N-terminus of MMP-3(DeltaC) 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 changes in hydrogen bonding near the active site of MMP-3(DeltaC) are reported. N-TIMP-1 binding causes the amide of Tyr223 of MMP-3(DeltaC) bound by N-TIMP-1 to exchange with water rapidly, implying a lack of the hydrogen bond observed in the crystal structure. The backbone amide proton of Asn162 becomes protected from rapid exchange upon forming a complex with N-TIMP-1 and could form a hydrogen bond to N-TIMP-1. N-TIMP-1 binding dramatically increases the rate of amide hydrogen exchange of Asp177 of the fifth beta strand of MMP-3(DeltaC), disrupting its otherwise stable hydrogen bond.
authors.affiliation: Department of Biochemistry, University of Missouri, Columbia 65211, USA.
authors.forename: S
authors.initials: S
authors.lastname: Arumugam
authors.forename: C L
authors.initials: CL
authors.lastname: Hemme
authors.forename: N
authors.initials: N
authors.lastname: Yoshida
authors.forename: K
authors.initials: K
authors.lastname: Suzuki
authors.forename: H
authors.initials: H
authors.lastname: Nagase
authors.forename: M
authors.initials: M
authors.lastname: Berjanskii
authors.forename: B
authors.initials: B
authors.lastname: Wu
authors.forename: S R
authors.initials: SR
authors.lastname: Van Doren
mesh_terms: D000577:Amides / Q000737:chemistry; D001665:Binding Sites; D004492:Edetic Acid; D005682:Gadolinium; D006801:Humans; D019278:Matrix Metalloproteinase 3 / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D015335:Molecular Probes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000577:Amides; D015335:Molecular Probes; D010446:Peptide Fragments; D011522:Protons; D011994:Recombinant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D004492:Edetic Acid; D005682:Gadolinium; D019278:Matrix Metalloproteinase 3