The high-resolution solution structure of the inhibitor-free catalytic fragment of human fibroblast collagenase (MMP-1), a protein of 18.7 kDa, 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 geometry-simulated annealing using a total of 3333 experimental NMR restraints, consisting of 2409 approximate interproton distance restraints, 84 distance restraints for 42 backbone hydrogen bonds, 426 torsion angle restraints, 125 3J(NHα) restraints, 153 Cα restraints, and 136 Cβ 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 Å for the backbone atoms, 0.80 ± 0.04 Å for all atoms, and 0.50 ± 0.03 Å for all atoms excluding disordered side chains. The overall structure of MMP-1 is composed of a β-sheet consisting of five β-strands in a mixed parallel and anti-parallel arrangement and three α-helices. A best-fit superposition of the NMR structure of inhibitor-free MMP-1 with the 1.56 Å 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) Proteins: Struct., Funct., Genet. 19, 98-109] complexed with a hydroxamate inhibitor yields a backbone atomic rms difference of 1.22 Å. 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 in mobility for residues 138-144 and a displacement for the Ca2+-loop (residues 74- 80). Distinct differences were observed for side-chain torsion angles, in particular, the ξ1 for N80 is -60°in the NMR structure compared to 180°in the X-ray. This results in the side chain of N80 occupying and partially blocking access tO the active site of MMP-1.
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