An NMR analysis of ubiquitin recognition by yeast ubiquitin hydrolase: evidence for novel substrate recognition by a cysteine protease.
Yeast ubiquitin hydrolase 1 (YUH1), a cysteine protease that catalyzes the removal of ubiquitin C-terminal adducts, is important for the generation of monomeric ubiquitin. Heteronuclear NMR spectroscopy has been utilized to map the YUH1 binding surface on ubiquitin. When YUH1 was titrated into a sample of ubiquitin, approximately 50% of the (1)H-(15)N correlation peaks of ubiquitin were affected to some degree, as a result of binding to YUH1. It is noteworthy that the amide resonances of the basic residues (Arg42, Lys48, Arg72, and Lys74) were highly perturbed. These positively charged basic residues may be involved in direct interactions with the negatively charged acidic residues on YUH1. In addition to the electrostatic surface, the hydrophobic surfaces on ubiquitin (Leu8, Ile44, Phe45, Val70, Leu71, and Leu73) and YUH1 are also likely to contribute to the binding interaction. Furthermore, the amide resonances of Ile13, Leu43, Leu50, and Leu69, the side chains of which are not on the surface, were also highly perturbed, indicating substrate-induced changes in the environments of these residues as well. These large changes, observed from residues located throughout the five-stranded beta-sheet surface and the C-terminus, suggest that substrate recognition by YUH1 involves a wider area on ubiquitin.
Sakamoto
T
T
Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194-8511, Japan.
Tanaka
T
T
Ito
Y
Y
Rajesh
S
S
Iwamoto-Sugai
M
M
Kodera
Y
Y
Tsuchida
N
N
Shibata
T
T
Kohno
T
T
D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013379:Substrate Specificity; D014452:Ubiquitins / Q000737:chemistry / Q000378:metabolism*
D014452:Ubiquitins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C059237:ubiquitin-Nalpha-protein hydrolase