Structural analysis of cysteine protease ubiquitin carboxy-terminal hydrolase UCHL1 in complex with the suicide inhibitor Z-VAE(OMe)-FMK

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a Parkinson's disease-associated cysteine hydrolase found abundantly and selectively expressed in neurons. The three dimensional structure of UCHL1 has previously been determined by x-ray diffraction at 2.4 Å resolution. The structure reveals that the catalytic residues (Cys 90, His 161, Asp 176) are misaligned, with nucleophilic cysteine (Cys 90) 7.7 Å away from the general base His 161, consistent with an inactive form of the enzyme. In this thesis, analysis of the co-crystal structure of UCHL1 bound to the suicide inhibitor, benzyloxycarbonyl-valine-alanine-glutamic-acid (OMe) fluoromethylketone (ZVAE(OMe)-FMK), is described. This structure reveals the mechanism of UCHL1 inactivation by the suicide inhibitor and allows a visualization of the intermolecular contacts between the enzyme and the inhibitor. Inspection of the co-crystal structure reveals that the Cys 90 of the enzyme is covalently linked to the inhibitor via a thioester bond. While the covalent bond allows tethering of the inhibitor to the active-site cysteine, a number of intermolecular interactions are observed between the inhibitor and the residues of the enzyme lining the S&feet; side of the active-site cleft. The structure also reveals that the Z-VAE(OMe)-FMK inhibitor is bound to the active site with the catalytic triad in an unproductive form, in which the general base histidine (His 161) is 8.0 Å away from the catalytic Cys 90, implying that the inhibitor may be expected to exhibit selectivity over other ubiquitin hydrolases, such as UCHL3, which have canonical catalytic triads.