Structural basis of substrate selection by ubiquitin carboxyl terminal hydrolases

The ubiquitin carboxyl terminal hydrolases (UCHs) are a family of cysteine proteases that catalyze the removal of adducts to the C-terminus of the ubiquitin. Ubiquitin is a very highly conserved protein in eukaryotic organisms where it functions, in part, as a signal targeting protein for proteolysis at the 26S protease. One product of the deubiquitinating reaction is ubiquitin with a C-terminal COOH, a feature necessary for activation of ubiquitin and conjugation to substrates. The work here describes the three dimensional structures of two UCHs determined by X-ray crystallography. One of these structures is of the unliganded human UCH-L3 enzyme, and the other is a complex of the yeast Yuhl enzyme bound to the substrate analogue ubiquitin aldehyde (Ubal). The UCH fold is comprised of a central antiparallel p-sheet which is packed against a-helices on both sides. This structure resembles that of the papain family of cysteine proteases; an observation that the UCH-L3 active site is comprised of a cysteine-histidine-aspartate catalytic triad and a glutamine oxyanion hole. The Yuhl-Ubal complex structure mimics the tetrahedral intermediate of the enzymatic reaction, with a covalent bond formed between the C-terminal carbon atom of Ubal and the active site cysteine of Yuhl. The interaction seen between Yuhl and Ubal, which is extensive and includes numerous hydrogen bonds and van der Waals interactions, explains the specificity of UCH enzymes for ubiquitin adducts. Differences between UCH-L3 and Yuhl-Ubal structures suggest that the enzyme active site is sequestered in the absence of substrate, and that the energy of binding ubiquitin is coupled to formation of an active conformation. One of the apparent conformational changes upon binding Ubal is the ordering of a loop tightly across the active-site cleft, such that substrates are probably required to orient their C-terminal adducts through a narrow opening.