Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 107, Issue 20, Pages 9117-9122Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0910870107
Keywords
deubiquitinating enzyme; enzyme suicide substrate complex; neurodegeneration; ubiquitination
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Funding
- National Cancer Institute [Y1-CO-1020]
- National Institute of General Medical Science [Y1-GM-1104]
- U.S. Department of Energy, Basic Energy Sciences, Office of Science [DE-AC02-06CH11357]
- Showalter grant
- Purdue University
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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 angstrom 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 angstrom 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 angstrom 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.
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