Ubiquitin vinyl methyl ester binding orients the misaligned active site of the ubiquitin hydrolase UCHL1 into productive conformation

Abstract

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 Å 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 β-hairpin at the distal site-a surface-exposed hydrophobic crevice 17 Å 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 Å 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.deubiquitinating enzyme | enzyme suicide substrate complex | neurodegeneration | ubiquitination U CHL1, a member of the UCH (ubiquitin C-terminal hydrolase) family of deubiquitinating enzymes (DUBs), is a 223-amino acid protein found abundantly and selectively expressed in brain, constituting up to 1-2% of total brain protein (1, 2). In vivo studies suggest that UCHL1 is involved in regulation of ubiquitin pool, apoptosis, and learning and memory, and its absence in mice because of spontaneous intragenic deletions yields phenotypes with neurological defects (3). Mutations in UCHL1 have been implicated in Parkinson disease (PD). A point mutation near the active site that changes Ile93 to Met (I93M) has been linked to an increased risk of developing an autosomaldominant form of PD (4). Conversely, a common S18Y polymorphism reduces susceptibility to PD (5, 6) and Alzheimer's disease (7). In addition to its association with neurodegenerative diseases, abnormal expression of UCHL1 is found in many forms of cancer, including lung, colorectal, and pancreatic cancers, and may be related to tumor progression (8, 9). The normal function of UCHL1, however, is not known. Also unknown are how the activity of this abundant neuronal enzyme is regulated and what its true physiological substrates are, although biochemical studies have indicated that UCHL1 can accept short-peptide (α or ϵ-amino-linked) or small-molecule C-terminal conjugates of ubiquitin as substrates, cleaving, as its name suggests, the amide bond following immediately after the C-terminal glycine (Gly7...

Recessive loss of function of the neuronal ubiquitin hydrolase UCHL1 leads to early-onset progressive neurodegeneration

“…To gain further insight into the role of UCHL1 in neurodegeneration, we examined the structural implications and functional consequences of the GLU7ALA mutation. The tertiary structure of UCHL1 resembles that of the papain family (12) and is characterized by the presence of a crossover loop, termed L8, that spans and thereby restricts access to the catalytic cleft that consists of the histidine-cysteine-aspartic acid triad (12,13) (Fig. 2A).…”

“…2A). In UCHL1, the L8 loop is short, thus requiring the ubiquitin substrate to tunnel underneath it to achieve proteolysis; residue E7 is located directly at the threshold of this tunnel (12,13). In the complex of UCHL1 and the ubiquitin substrate mimic, ubiquitin vinyl methyl ester (UbVMe) (13), GLU7 is involved in a hydrogen-bonding network that interacts both with the ubiquitin substrate mimic and the L8 loop ( Fig.…”

“…We next analyzed the effects of the GLU7ALA mutation on ubiquitin binding and C-terminal hydrolase enzymatic activity. In Escherichia coli, we expressed and purified WT UCHL1 (UCHL1 WT ) and mutants UCHL1 GLU7ALA , UCHL1 ILE93MET , and UCHL1 CYS90SER (a cysteine to serine substitution in position 90), which carries a mutation in the active cleft consisting of the abovementioned triad and was previously shown to interfere with the catalytic activity (12)(13)(14). First, the binding of ubiquitin to these purified proteins was measured using isothermal titration calorimetry, from which the K d values for ubiquitin binding to these proteins were calculated ( Fig.…”

Characterization of the Folding of a 5 2 -Knotted Protein Using Engineered Single-Tryptophan Variants

“…In all cases, the structures and activities of the variants are very similar to those of the WT. This indicates that the partially exposed positions selected for the incorporation of Trp are not involved in either substrate binding or the subsequent conformational change that is required for activity by this enzyme (26,41).…”

Using Protein Motion to Read, Write, and Erase Ubiquitin Signals

“…Proper arrangement of the catalytic residues can be accomplished in multiple ways. UCHL1 is activated by ubiquitin itself, such that binding of the globular portion of ubiquitin realigns the enzyme's active site around ubiquitin's C terminus (89). The catalytic center of USP7 also rearranges in the presence of ubiquitin to both tighten around ubiquitin's C-terminal tail and form a competent active site (88).…”