THE STRUCTURE OF HUMAN CATHEPSIN-L AT 2Å RESOLUTION. Richard Pauptit, Alec Tucker*, Simon Weston & Bob Gordon^. Zeneca Pharmaceuticals, Alderley Park, Macclesfield UK. *Pfizer Central Research, Sandwich, Kent, UK ^Jannsen, Antwerp, Belgium.

Cathepsin L is a thiol protease which plays an important role in protein degradation within the lysozymes and in the extracellular matrix, and is a leading target in the search for inhibitors of bone resorption. Human cathepsin L has been cloned, expressed in E.coli, purified, crystallised, and its three-dimensional structure has been determined by molecular replacement methods using papain as a trial model. Purification was problematic, but once 0.1 mm crystals were obtained structure solution progressed rapidly. The crystals froze readily since they were grown in 30% MPD. They are P21 with a=46.23, b=49.38, c=49.25 Å and beta=113.45 deg. The overall structure is very similar to other thiol proteases; in particular, the active site is similar to papain suggesting equivalent mechanism of action. However, there are differences in detail of chain tracing. Close to the active site in the region where inhibitors bind there are differences in the arrangement of hydrohobic sidechains, suggesting the design of specific inhibitors is feasible. The active site thiol group is oxidised, and the active site cleft contains an unidentified peptide-like fragment. While cathepsin L is biosynthesised as a pre-pro form of 333 residues and loses a signal sequence to give a 316 residue pro-form which is cleaved at low pH to yield a 219 residue form, the crystals contain a further-cleaved 2-chain form of 175 and 42 residues which are covalently linked through a disulfide bond. The solvent contains some MPD molecules, over 150 water molecules, and, apparently, cacodylate ions. The structure was refined to R=18.4% at 2Å resolution.