CRYSTAL-STRUCTURES OF RECOMBINANT RAT CATHEPSIN-B AND A CATHEPSIN B-INHIBITOR COMPLEX - IMPLICATIONS FOR STRUCTURE-BASED INHIBITOR DESIGN

The lysosomal cysteine proteinase cathepsin B (EC 3.4.22.1) plays an i mportant role in protein catabolism and has also been implicated in va rious disease states. The crystal structures of two forms of native re combinant rat cathepsin B have been determined. The overall folding of rat cathepsin B was shown to be very similar to that of the human liv er enzyme. The structure of the native enzyme containing an underivati zed active site cysteine (Cys(29)) showed the active enzyme conformati on to be similar to that determined previously for the oxidized form. In a second structure Cys(29) was derivatized with the reversible bloc king reagent pyridyl disulfide. In this structure large side chain con formational changes were observed for the two key catalytic residues C ys(29) and His(199), demonstrating the potential flexibility of these side chains. In addition the structure of the complex between rat cath epsin B and the inhibitor benzyloxycarbonyl-Arg-Ser(O-Bzl) chloromethy lketone was determined. The complex structure showed that very little conformational change occurs in the enzyme upon inhibitor binding. It also allowed visualization of the interaction between the enzyme and i nhibitor, In particular the interaction between Glu(245) and the P-2 A rg residue was clearly demonstrated, and it was found that the benzyl group of the P-1 substrate residue occupies a large hydrophobic pocket thought to represent the S'(1) subsite, This may have important impli cations for structure-based design of cathepsin B inhibitors.