NMR Mapping and Functional Confirmation of the Collagen Binding Sites of MMP-2

• Published in: Biochemistry (Easton) Vol. 48; no. 25; pp. 5822 - 5831
• Main Authors: Xu, Xiaoping, Mikhailova, Margarita, Ilangovan, Udayar, Chen, Zhihua, Yu, Agnes, Pal, Sanjay, Hinck, Andrew P., Steffensen, Bjorn
• Format: Journal Article
• Language: English
• Published: 30.06.2009
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi900513h

Interactions of matrix metalloproteinase-2 (MMP-2) 1 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 an CBD binding site on the α1(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 shifts perturbations of several surface exposed CBD backbone amide resonances in a concentration dependent manner. The ten residues that underwent the largest chemical shift perturbations (R 252 in module 1, R 296 , F 297 , Y 302 , E 321 , Y 323 , 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 1D 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 binding of CBD to native and denatured type I collagen and demonstrated that all three modules contribute to the 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.