CRYSTAL STRUCTURES OF STROMELYSIN: CONFORMATIONAL CHANGES INDUCED BY INHIBITOR BINDING. Longyin Chen, Timothy J. Rydel, C. Michelle Dunaway, Larry Strickland, The Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, OH 45253-8707

Stromelysin, a zinc-dependent proteinase, is a member of the matrix metalloproteinases (MMP). MMPs are believed to play an important role in pathological conditions such as osteoarthritis, rheumatoid arthritis and tumor invasion. The crystal structures of native truncated stromelysin (the catalytic domain) and its inhibitor-bound complexes have been solved. The native structure has been refined to 2.0 Å resolution (R-factor 0.21, R-free 0.29). The complexes were formed both from diffusion soaking and co-crystallization, and have been refined to 2.3 Å resolution (R-factor and R-free 0.19 and 0.31 for the soaked crystals; 0.19 and 0.33 for the co-crystals, respectively). The native and the inhibitor-soaked crystals are in space group P2₁2₁2₁, with two molecules (A and B) in the asymmetric unit, associating as a dimer. The inhibitor-bound co-crystals, also existing as a dimer, are in space group C2, one molecule per asymmetric unit. There are three [[alpha]]-helices and one twisted, five-strand [[beta]]-sheet in each molecule with one catalytic Zn²⁺, one structural Zn²⁺ and three Ca²⁺ ions. The catalytic zinc is ligated to three histidines (H201, H205 and H211), with distances around 2.1-2.2 Å. The fourth and possibly the fifth ligands may come from substrate, inhibitor, another stromelysin molecule or solvent.

The inhibitor binding obtained by soaking or co-crystallization is basically identical. The Pl' portion of the inhibitor binds to a highly hydrophobic pocket. Most residues between the native and the inhibitor-bound complex structures match very well except the inhibitor-binding loop (residues 222-231). The major difference occurs at Y223. In native structure, the side chain of Y223 occupies the position that is occupied by the P1' portion of an inhibitor in the complex. The inhibitor has to push away the Y223 in order to fit into the binding site. Moreover, the inhibitor binding and Y223 movement induce movement of the entire loop. This makes the loop completely different in the native and in the complex. Some residues (C[[alpha]]) differ by as much as 5 Å. In native stromelysin, a symmetry-related molecule B has its C-terminal (251-255) interacting with the active site of molecule A of the central unit. The last residue, T255, has its carboxylate group ligated to the catalytic zinc, forming two ligand bonds, in a similar manner as an inhibitor. This symmetrical C-terminal-active site interaction occurs for one molecule only. In molecule A, the last four C-terminal residues are disordered as a flexible tail. The situation in the inhibitor-bound complexes is more complicated. In molecule B, only the inhibitor binds to the active site. But in molecule A, inhibitor competes with the C-terminal of a symmetry-related molecule B.