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 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 15N-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 (R252 in module 1, R296, F297, Y302, E321, Y323, and Y329 in module 2, and R368, W374, and Y381 in module 3) were investigated by site-specific substitution with alanine. The structural integrity of the CBD variants was also analyzed by one-dimensional 1H 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.
X. Xu, Mikhailova, M., Ilangovan, U., Chen, Z., Yu, A., Dr. Sanjay Pal, Hinck, A. P., and Steffensen, B., “Nuclear magnetic resonance mapping and functional confirmation of the collagen binding sites of matrix metalloproteinase-2”, Biochemistry, vol. 48, pp. 5822–5831, 2009.