Ubiquitin vinyl methyl ester binding orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive conformation.
Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a Parkinson disease-associated, putative cysteine protease found abundantly and selectively expressed in neurons. The crystal structure of apo UCHL1 showed that the active-site residues are not aligned in a canonical form, with the nucleophilic cysteine being 7.7 A from the general base histidine, an arrangement consistent with an inactive form of the enzyme. Here we report the crystal structures of the wild type and two Parkinson disease-associated variants of the enzyme, S18Y and I93M, bound to a ubiquitin-based suicide substrate, ubiquitin vinyl methyl ester. These structures reveal that ubiquitin vinyl methyl ester binds primarily at two sites on the enzyme, with its carboxy terminus at the active site and with its amino-terminal beta-hairpin at the distal site-a surface-exposed hydrophobic crevice 17 A away from the active site. Binding at the distal site initiates a cascade of side-chain movements in the enzyme that starts at a highly conserved, surface-exposed phenylalanine and is relayed to the active site resulting in the reorientation and proximal placement of the general base within 4 A of the catalytic cysteine, an arrangement found in productive cysteine proteases. Mutation of the distal-site, surface-exposed phenylalanine to alanine reduces ubiquitin binding and severely impairs the catalytic activity of the enzyme. These results suggest that the activity of UCHL1 may be regulated by its own substrate.
Boudreaux
David A
DA
Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA.
Maiti
Tushar K
TK
Davies
Christopher W
CW
Das
Chittaranjan
C
D002151:Calorimetry; D020134:Catalytic Domain / Q000235:genetics / Q000502:physiology*; D003460:Crystallization; D004926:Escherichia coli; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D043222:Ubiquitin Thiolesterase / Q000737:chemistry* / Q000378:metabolism; D014452:Ubiquitins / Q000737:chemistry* / Q000378:metabolism
C482860:UCHL1 protein, human; D014452:Ubiquitins; C496729:ubiquitin vinylmethyl ester; D043222:Ubiquitin Thiolesterase