High-resolution solution structure of the inhibitor-free catalytic fragment of human fibroblast collagenase determined by multidimensional NMR. Biochemistry;37:1495–1504

ABSTRACT: 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 3 J NHR restraints, 153 CR restraints, and 136 C 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 -sheet consisting of five -strands in a mixed parallel and anti-parallel arrangement and three R-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 Ca 2+ -loop (residues 74-80). Distinct differences were observed for side-chain torsion angles, in particular, the 1 for N80 is -60°in the NMR structure compared to 180°i n the X-ray. This results in the side chain of N80 occupying and partially blocking access to the active site of MMP-1. The matrix metalloproteinase (MMP) 1 family, which includes the collagenases, stromelysins, and gelatinases, is involved in the degradation of the extracellular matrix which is associated with normal tissue remodeling processes such as pregnancy, wound healing, and angiogenesis (2-4). These enzymes are modular with both propeptide and catalytic domains being common to the entire family (5, 6) while requiring both zinc and calcium for catalytic activity (7-9). MMP expression and activity is highly controlled because of the degradative nature of these enzymes. The MMPs are regulated by either specific inhibitors (tissue inhibitor of metalloendoproteases, TIMP), by cleavage of the inactive proenzyme or by transcription induction or suppression (3). A number of biochemical stimuli including cytokines, hormones, oncogene products and tumor promoters also effect the synthesis and activation of The high-resolution structure of an enzyme free of either ligand or inhibitor provides an initial framework from which a structure-based drug development program is established. The rational for this approach is that future NMR refinements and analysis of enzyme-ligand complexes are determined by difference to enzyme-free NMR spectra. Additionally, valuable structural information may also be obtained from understanding any conformational change induced in the enzyme upon binding an inhibitor. Therefore, a structural program to determine the high-resolution NMR solution structure of the inhibitor-free catalytic fragment of human fibroblast collagenase was initiated. In a previous paper (13), we presented the near complete 1 H, 15 N, 13 CO, and 13 C assignments, solution secondary structure, and dynamics for MMP-1 which comprise the essential foundation for such a study. In this paper, we present the determination of a highresolution solution structure of inhibitor-free MMP-1 using three-dimensional heteronuclear NMR spectroscopy. The resulting high-resolution solution structure is based on a total ‡ Atomic coordinates for the 30 final simulated annealing structures and the restrained minimized mean structure of MMP-1 have been deposited in the Brookhaven Protein Data Bank (PDB ID code 1ayk and 2ayk, respectfully)