STRUCTURE OF RECOMBINANT HUMAN CPP32 IN COMPLEX WITH THE TETRAPEPTIDEACETYL-ASP-VAL-ALA-ASP FLUOROMETHYL KETONE

The cysteine protease CPP32 has been expressed in a soluble form in Es cherichia coli and purified to >95% purity. The three-dimensional stru cture of human CPP32 in complex with the irreversible tetrapeptide inh ibitor acetyl Asp Val-Ala-Asp fluoromethyl ketone was determined by x- ray crystallography at a resolution of 2.3 Angstrom. The asymmetric un it contains a (p17/p12)(2) tetramer, in agreement with the tetrameric structure of the protein in solution as determined by dynamic light sc attering and size exclusion chromatography. The overall topology of CP P32 is very similar to that of interleukin-1 beta-converting enzyme (I CE); however, differences exist at the N terminus of the p17 subunit, where the first helix found in ICE is missing in CPP32. A deletion/ins ertion pattern is responsible for the striking differences observed in the loops around the active site. In addition, the P1 carbonyl of the ketone inhibitor is pointing into the oxyanion hole and forms a hydro gen bond with the peptidic nitrogen of Gly-122, resulting in a differe nt state compared with the tetrahedral intermediate observed in the st ructure of ICE and CPP32 in complex with an aldehyde inhibitor, The to pology of the interface formed by the two p17/p12 heterodimers of CPP3 2 is different from that of ICE. This results in different orientation s of CPP32 heterodimers compared with ICE heterodimers, which could af fect substrate recognition. This structural information will be invalu able for the design of small synthetic inhibitors of CPP32 as well as for the design of CPP32 mutants.