The binding site for UCH-L3 on ubiquitin: Mutagenesis and NMR studies on the complex between ubiquitin and UCH-L3

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 protei n to the proteasome, the nucleus, the cytoskeleton or the endocytotic machi nery. Recognition of the ubiquitin domain by a variety of enzymes and recep tors is vital to the targeting function of ubiquitin. Several parallel path ways exist and these must be able to distinguish among ubiquitin, several d ifferent types of polymeric ubiquitin, and the various ubiquitin-like domai ns. Here we report the first molecular description of the binding site on u biquitin for ubiquitin C-terminal hydrolase L3 (UCH-L3). The site on ubiqui tin was experimentally determined using solution NMR, and site-directed mut agenesis. The site on UCH-L3 was modeled based on X-ray crystallography, mu ltiple sequence alignments, and computer-aided docking. Basic residues loca ted on ubiquitin (K6, K11, R72, and R74) are postulated to contact acidic r esidues on UCH-LS (E10, E14, D33, E219). These putative interactions are te stable and fully explain the selectivity of ubiquitin domain binding to thi s enzyme. (C) 1999 Academic Press.