title: Nuclear magnetic resonance mapping and functional confirmation of the collagen binding sites of matrix metalloproteinase-2.
abstract: Interactions of matrix metalloproteinase-2 (MMP-2) with native and denatured forms of several types of collagen are mediated by the collagen binding domain (CBD). CBD positions substrates relative to the catalytic site and is essential for their cleavage. Our previous studies identified a CBD binding site on the alpha1(I) collagen chain. The corresponding synthetic collagen peptide P713 bound CBD with high affinity and was used in this study to identify specific collagen binding residues by NMR analysis of (15)N-labeled CBD complexed with P713. Results obtained showed that P713 caused chemical shift perturbations of several surface-exposed CBD backbone amide resonances in a concentration-dependent manner. The 10 residues that underwent the largest chemical shift perturbations (R(252) in module 1, R(296), F(297), Y(302), E(321), Y(323), and Y(329) in module 2, and R(368), W(374), and Y(381) in module 3) were investigated by site-specific substitution with alanine. The structural integrity of the CBD variants was also analyzed by one-dimensional (1)H NMR. Surface plasmon resonance and microwell protein binding assays of control and CBD variants showed that residues in all three CBD modules contributed to collagen binding. Single-residue substitutions altered the affinity for peptide P713, as well as native and denatured type I collagen, with the greatest effects observed for residues in modules 2 and 3. Additional alanine substitutions involving residues in two or three modules simultaneously further reduced the level of binding of CBD to native and denatured type I collagen and demonstrated that all three modules contribute to substrate binding. These results have localized and confirmed the key collagen binding site residues in the three fibronectin type II-like modules of MMP-2.
authors.affiliation: Department of Periodontics, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, MC 7894, San Antonio, Texas 78229-3900, USA.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Xu
authors.forename: Margarita
authors.initials: M
authors.lastname: Mikhailova
authors.forename: Udayar
authors.initials: U
authors.lastname: Ilangovan
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Chen
authors.forename: Agnes
authors.initials: A
authors.lastname: Yu
authors.forename: Sanjay
authors.initials: S
authors.lastname: Pal
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Hinck
authors.forename: Bjorn
authors.initials: B
authors.lastname: Steffensen
mesh_terms: D001665:Binding Sites / Q000235:genetics; D003094:Collagen / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D005353:Fibronectins / Q000737:chemistry / Q000145:classification; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D040681:Structural Homology, Protein
chemical_list: D005353:Fibronectins; D003094:Collagen; D020778:Matrix Metalloproteinase 2