Ubiquitin Binding Interface Mapping on Yeast Ubiquitin Hydrolase by NMR Chemical Shift Perturbation
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- 作者：Sundaresan Rajesh ; Taiichi Sakamoto ; Mariko Iwamoto-Sugai ; Takehiko Shibata ; Toshiyuki Kohno ; and Yutaka Ito
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：July 20, 1999
- 年：1999
- 卷：38
- 期：29
- 页码：9242 - 9253
- 全文大小：276K
- 年卷期：v.38,no.29(July 20, 1999)
- ISSN：1520-4995
文摘
The interaction between the 26 kDa yeast ubiquitin hydrolase (YUH1), involved in maintainingthe monomeric ubiquitin pool in cells, and the 8.5 kDa yeast ubiquitin protein has been studied byheteronuclear multidimensional NMR spectroscopy. Chemical shift perturbation of backbone 1HN, 15N,and 13C
resonances of YUH1, in a YUH1-ubiquitin mixture and in a 35 kDa covalent complex withubiquitin (a stable analogue of the tetrahedral reaction intermediate), was employed to identify the ubiquitinbinding interface of YUH1. This interface mapped on the secondary structure of YUH1 suggests a widearea of contact for ubiquitin, encompassing the N-terminus, 1, 4, 
2, 3, and 6, coincident with thehigh specificity of YUH1 for ubiquitin. The presence of several hydrophobic clusters in the ubiquitinbinding interface of YUH1 suggests that hydrophobic interactions are equally important as ionic interactionsin contacting ubiquitin. The residues in the binding interface exhibit a high percentage of homology amongthe members of the ubiquitin C-terminal hydrolase family, indicating the well-conserved nature of theubiquitin binding interface reported in this study. The secondary structure of YUH1, from our NMRstudies, was similar to the recently determined structure of its human homologue ubiquitin C-terminalhydrolase L3 (UCH-L3), except for the absence of the helix H3 of UCH-L3. This region in YUH1 (helixH3 of UCH-L3) was least perturbed upon ubiquitin binding. Therefore, the binding interface was mappedonto the corresponding residues in the UCH-L3 crystal structure. A model for ubiquitin binding to YUH1is proposed, in which a good correlation was observed for the lateral binding of ubiquitin to UCH-L3(YUH1), stabilized by the electrostatic and hydrophobic interactions.
resonances of YUH1, in a YUH1-ubiquitin mixture and in a 35 kDa covalent complex withubiquitin (a stable analogue of the tetrahedral reaction intermediate), was employed to identify the ubiquitinbinding interface of YUH1. This interface mapped on the secondary structure of YUH1 suggests a widearea of contact for ubiquitin, encompassing the N-terminus, 1, 4, 2, 3, and 6, coincident with thehigh specificity of YUH1 for ubiquitin. The presence of several hydrophobic clusters in the ubiquitinbinding interface of YUH1 suggests that hydrophobic interactions are equally important as ionic interactionsin contacting ubiquitin. The residues in the binding interface exhibit a high percentage of homology amongthe members of the ubiquitin C-terminal hydrolase family, indicating the well-conserved nature of theubiquitin binding interface reported in this study. The secondary structure of YUH1, from our NMRstudies, was similar to the recently determined structure of its human homologue ubiquitin C-terminalhydrolase L3 (UCH-L3), except for the absence of the helix H3 of UCH-L3. This region in YUH1 (helixH3 of UCH-L3) was least perturbed upon ubiquitin binding. Therefore, the binding interface was mappedonto the corresponding residues in the UCH-L3 crystal structure. A model for ubiquitin binding to YUH1is proposed, in which a good correlation was observed for the lateral binding of ubiquitin to UCH-L3(YUH1), stabilized by the electrostatic and hydrophobic interactions.