Cell Cycle and Cancer Unit, St. Vincent's Institute of Medical Research, Melbourne, VIC Australia.
Cell Cycle. 2013 Jun 1;12(11):1732-44. doi: 10.4161/cc.24818. Epub 2013 May 8.
The attachment of ubiquitin (Ub) to lysines on substrates or itself by ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes results in protein ubiquitination. Lysine selection is important for generating diverse substrate-Ub structures and targeting proteins to different fates; however, the mechanisms of lysine selection are not clearly understood. The positioning of lysine(s) toward the E2/E3 active site and residues proximal to lysines are critical in their selection. We investigated determinants of lysine specificity of the ubiquitin-conjugating enzyme Cdc34, toward substrate and Ub lysines. Evaluation of the relative importance of different residues positioned -2, -1, +1 and +2 toward ubiquitination of its substrate, Sic1, on lysine 50 showed that charged residues in the -1 and -2 positions negatively impact on ubiquitination. Modeling suggests that charged residues at these positions alter the native salt-bridge interactions in Ub and Cdc34, resulting in misplacement of Sic1 lysine 50 in the Cdc34 catalytic cleft. During polyubiquitination, Cdc34 showed a strong preference for Ub lysine 48 (K48), with lower activity towards lysine 11 (K11) and lysine 63 (K63). Mutating the -2, -1, +1 and +2 sites surrounding K11 and K63 to mimic those surrounding K48 did not improve their ubiquitination, indicating that further determinants are important for Ub K48 specificity. Modeling the ternary structure of acceptor Ub with the Cdc34~Ub complex as well as in vitro ubiquitination assays unveiled the importance of K6 and Q62 of acceptor Ub for Ub K48 polyubiquitination. These findings provide molecular and structural insight into substrate lysine and Ub K48 specificity by Cdc34.
泛素（Ub）与连接酶（E2）和泛素连接酶（E3）在底物赖氨酸或自身上的连接导致蛋白质泛素化。赖氨酸的选择对于产生不同的底物-Ub 结构和将蛋白质靶向不同命运至关重要；然而，赖氨酸选择的机制尚不清楚。赖氨酸（s）向 E2/E3 活性位点的定位以及赖氨酸附近的残基对于它们的选择至关重要。我们研究了泛素连接酶 Cdc34 对底物和 Ub 赖氨酸的赖氨酸特异性决定因素。评估不同残基在位置 -2、-1、+1 和+2 时对其底物 Sic1 上赖氨酸 50 的泛素化的相对重要性表明，-1 和-2 位置的带电残基对泛素化有负面影响。建模表明，这些位置的带电残基改变了 Ub 和 Cdc34 中的天然盐桥相互作用，导致 Sic1 赖氨酸 50 在 Cdc34 催化裂缝中的错位。在多泛素化过程中，Cdc34 对 Ub 赖氨酸 48（K48）表现出强烈的偏好，对赖氨酸 11（K11）和赖氨酸 63（K63）的活性较低。突变围绕 K11 和 K63 的 -2、-1、+1 和+2 位点以模拟围绕 K48 的位点并没有提高它们的泛素化，这表明进一步的决定因素对于 Ub K48 的特异性很重要。对接受 Ub 的三元结构与 Cdc34~Ub 复合物以及体外泛素化测定进行建模揭示了接受 Ub 的 K6 和 Q62 对 Ub K48 多泛素化的重要性。这些发现为 Cdc34 对底物赖氨酸和 Ub K48 特异性提供了分子和结构上的见解。
