Identification of a substrate recognition site on Ubc9
Identification of a substrate recognition site on Ubc9
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DOI：
10.1074/jbc.m108418200
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发表时间：
2002-06-14
影响因子：
4.8
通讯作者：
Chen, Y
中科院分区：
文献类型：
--
作者：
Lin, DH;Tatham, MH;Chen, Y
人Ubc 9与泛素结合酶同源。然而，它不是缀合泛素，而是缀合泛素同源物，小泛素样修饰物1（SUMO-1），也称为UBL 1、GMP 1、SMTP 3、PIC 1和sentrin。SUMO-1缀合途径在泛素活化酶（El）的一级序列、泛素缀合酶（E2）的三维结构和整个缀合途径的化学性质方面与泛素的缀合途径非常相似。使用NMR光谱研究了底物与Ubc 9的相互作用。具有与来自p53和c-Jun的SUMO-1缀合位点的序列相对应的序列的肽都结合至与人Ubc 9的活性位点Cys（93）相邻的表面，所述活性位点Cys（93）先前已显示包括在微秒至毫秒时间尺度上表现出最显著动力学的残基。构建该区域中的突变Q126 A、Q130 A、A131 D、E132 A、Y134 A和T135 A，以评价这些残基在SUMO-1缀合中的作用。这些改变对SUMO-1与靶蛋白p53、E1 B和早幼粒细胞白血病蛋白的结合具有显著影响，并在Ubc 9上定义了底物结合位点。此外，p53的SUMO-1缀合位点在游离或与Ubc 9结合时不形成任何确定的二级结构。这表明在靶蛋白中SUMO-1缀合位点处的确定的二级结构对于SUMO-1途径的识别和缀合不是必需的。
Human Ubc9 is homologous to ubiquitin-conjugating enzymes. However, instead of conjugating ubiquitin, it conjugates a ubiquitin homologue, small ubiquitin-like modifier 1 (SUMO-1), also known as UBL1, GMP1, SMTP3, PIC1, and sentrin. The SUMO-1 conjugation pathway is very similar to that of ubiquitin with regard to the primary sequences of the ubiquitin-activating enzymes (El), the three-dimensional structures of the ubiquitin-conjugating enzymes (E2), and the chemistry of the overall conjugation pathway. The interaction of substrates with Ubc9 has been studied using NMR spectroscopy. Peptides with sequences that correspond to those of the SUMO-1 conjugation sites from p53 and c-Jun both bind to a surface adjacent to the active site Cys(93) of human Ubc9, which has been previously shown to include residues that demonstrate the most significant dynamics on the microsecond to millisecond time scale. Mutations in this region, Q126A, Q130A, A131D, E132A, Y134A, and T135A, were constructed to evaluate the role of these residues in SUMO-1 conjugation. These alterations have significant effects on the conjugation of SUMO-1 with the target proteins p53, E1B, and promyelocytic leukemia protein and define a substrate binding site on Ubc9. Furthermore, the SUMO-1 conjugation site of p53 does not form any defined secondary structure when either free or bound to Ubc9. This suggests that a defined secondary structure at SUMO-1 conjugation sites in target proteins is not necessary for recognition and conjugation by the SUMO-1 pathway.