Ubiquitin binding interface mapping on yeast ubiquitin hydrolase by NMR chemical shift perturbation
Ubiquitin binding interface mapping on yeast ubiquitin hydrolase by NMR chemical shift perturbation
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DOI：
10.1021/bi9903953
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发表时间：
1999-07-20
期刊：
影响因子：
2.9
通讯作者：
Ito, Y
中科院分区：
文献类型：
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作者：
Rajesh, S;Sakamoto, T;Ito, Y
用异核多维核磁共振技术研究了参与维持细胞内单体泛素库的26 kDa酵母泛素水解酶(YUH1)与8.5 kDa酵母泛素蛋白之间的相互作用。主链H-1(N)的化学位移微扰。在YUH1-泛素混合物和35 kDa的泛素共价络合物(四面体反应中间体的稳定类似物)中，YUH1的N-15和C-13(α)共振被用来鉴定YUH1的泛素结合界面。绘制在YUH1二级结构上的这个界面表明泛素有一个广泛的接触区域，包括N-末端、α1、α4、β2、β3和β6，这与YUH1对泛素的高度特异性相一致。在YUH1的泛素结合界面上存在几个疏水团簇，这表明在接触泛素时，疏水相互作用与离子相互作用同等重要。结合界面中的残基在泛素C末端水解酶家族成员中显示出很高的同源性，表明本研究中报道的泛素结合界面的保守性很好。根据我们的核磁共振研究，YUH1的二级结构与最近确定的其人类同源物泛素C末端水解酶L3(UCH-L3)的结构相似，只是UCH-L3没有螺旋H3。YUH1(UCH-L3的H3螺旋)的这个区域对泛素结合的干扰最小。因此，结合界面被映射到UCH-L3晶体结构中相应的残基上。提出了泛素与UCH-L3(YUH1)结合的模型，该模型通过静电和疏水相互作用稳定了泛素与UCH-L3(YUH1)的侧向结合。
The interaction between the 26 kDa yeast ubiquitin hydrolase (YUH1), involved in maintaining the monomeric ubiquitin pool in cells, and the 8.5 kDa yeast ubiquitin protein has been studied by heteronuclear multidimensional NMR spectroscopy. Chemical shift perturbation of backbone H-1(N). N-15, and C-13(alpha) resonances of YUH1, in a YUH1-ubiquitin mixture and in a 35 kDa covalent complex with ubiquitin (a stable analogue of the tetrahedral reaction intermediate), was employed to identify the ubiquitin binding interface of YUH1. This interface mapped on the secondary structure of YUH1 suggests a wide area of contact for ubiquitin, encompassing the N-terminus, alpha 1, alpha 4, beta 2, beta 3, and beta 6, coincident with the high specificity of YUH1 for ubiquitin. The presence of several hydrophobic clusters in the ubiquitin binding interface of YUH1 suggests that hydrophobic interactions are equally important as ionic interactions in contacting ubiquitin. The residues in the binding interface exhibit a high percentage of homology among the members of the ubiquitin C-terminal hydrolase family, indicating the well-conserved nature of the ubiquitin binding interface reported in this study. The secondary structure of YUH1, from our NMR studies, was similar to the recently determined structure of its human homologue ubiquitin C-terminal hydrolase L3 (UCH-L3), except for the absence of the helix H3 of UCH-L3. This region in YUH1 (helix H3 of UCH-L3) was least perturbed upon ubiquitin binding. Therefore, the binding interface was mapped onto the corresponding residues in the UCH-L3 crystal structure. A model for ubiquitin binding to YUH1 is 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.