HIGH-RESOLUTION SOLUTION STRUCTURE OF THE INHIBITOR-FREE CATALYTIC FRAGMENT OF HUMAN FIBROBLAST COLLAGENASE DETERMINED BY MULTIDIMENSIONAL NMR

The high-resolution solution structure of the inhibitor-free catalytic fragment of human fibroblast collagenase (MMP-1), a protein of 18.7 k Da, which is a member of the matrix metalloproteinase family, has been determined using three-dimensional heteronuclear NMR spectroscopy. A total of 30 structures were calculated by means of hybrid distance geo metry-simulated annealing using a total of 3333 experimental NMR restr aints, consisting of 2409 approximate interproton distance restraints, 84 distance restraints for 42 backbone hydrogen bonds, 426 torsion an gle restraints, 125 (3)J(NH alpha) restraints, 153 C alpha restraints, and 136 C beta restraints. The atomic rms distribution about the mean coordinate positions for the 30 structures for residues 7-137 and 145 -163 is 0.42 +/- 0.04 Angstrom for the backbone atoms, 0.80 +/- 0.04 A ngstrom for all atoms, and 0.50 +/- 0.03 Angstrom for all atoms exclud ing disordered side chains. The overall structure of MMP-1 is composed of a beta-sheet consisting of five beta-strands in a mixed parallel a nd anti-parallel arrangement and three alpha-helices. A best-fit super position of the NMR structure of inhibitor-free MMP-1 with the 1.56 An gstrom resolution X-ray structure by Spurlino et al. [Spurlino, J. C., Smallwood, A. M., Carlton, D. D., Banks, T. M., Vavra, K. J., Johnson , J. S., Cook, E. R., Falvo, J., and Wahl, R. C., et al. (1994) Protei ns: Struct., Funct., Genet. 19, 98-109] complexed with a hydroxamate i nhibitor yields a backbone atomic rms difference of 1.22 Angstrom. The majority of differences between the NMR and X-ray structure occur in the vicinity of the active site for MMP-1. This includes an increase i n mobility for residues 138-144 and a displacement for the Ca2+-loop ( residues 74-80). Distinct differences were observed for side-chain tor sion angles, in particular, the chi(1) for N80 is -60 degrees in the N MR structure compared to 180 degrees in the X-ray. This results in the side chain of N80 occupying and partially blocking access to the acti ve site of MMP-1.