title: The binding site for UCH-L3 on ubiquitin: mutagenesis and NMR studies on the complex between ubiquitin and UCH-L3.
abstract: The ubiquitin fold is a versatile and widely used targeting signal that is added post-translationally to a variety of proteins. Covalent attachment of one or more ubiquitin domains results in localization of the target protein to the proteasome, the nucleus, the cytoskeleton or the endocytotic machinery. Recognition of the ubiquitin domain by a variety of enzymes and receptors is vital to the targeting function of ubiquitin. Several parallel pathways exist and these must be able to distinguish among ubiquitin, several different types of polymeric ubiquitin, and the various ubiquitin-like domains. Here we report the first molecular description of the binding site on ubiquitin for ubiquitin C-terminal hydrolase L3 (UCH-L3). The site on ubiquitin was experimentally determined using solution NMR, and site-directed mutagenesis. The site on UCH-L3 was modeled based on X-ray crystallography, multiple sequence alignments, and computer-aided docking. Basic residues located on ubiquitin (K6, K11, R72, and R74) are postulated to contact acidic residues on UCH-L3 (E10, E14, D33, E219). These putative interactions are testable and fully explain the selectivity of ubiquitin domain binding to this enzyme.
authors.affiliation: Department of Biochemistry, Emory University, Atlanta, GA 30322, USA.
authors.forename: K D
authors.initials: KD
authors.lastname: Wilkinson
authors.forename: E
authors.initials: E
authors.lastname: Laleli-Sahin
authors.forename: J
authors.initials: J
authors.lastname: Urbauer
authors.forename: C N
authors.initials: CN
authors.lastname: Larsen
authors.forename: G H
authors.initials: GH
authors.lastname: Shih
authors.forename: A L
authors.initials: AL
authors.lastname: Haas
authors.forename: S T
authors.initials: ST
authors.lastname: Walsh
authors.forename: A J
authors.initials: AJ
authors.lastname: Wand
mesh_terms: D000495:Allosteric Site; D000595:Amino Acid Sequence; D003198:Computer Simulation; D017124:Conserved Sequence / Q000235:genetics; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010206:Papain / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D016415:Sequence Alignment; D055672:Static Electricity; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase; D014452:Ubiquitins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D014452:Ubiquitins; D013869:Thiolester Hydrolases; D043222:Ubiquitin Thiolesterase; D010206:Papain