Zhang Naixia, Wang Qinghua, Ehlinger Aaron, Randles Leah, Lary Jeffrey W, Kang Yang, Haririnia Aydin, Storaska Andrew J, Cole James L, Fushman David, Walters Kylie J
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, 55455, USA.
Mol Cell. 2009 Aug 14;35(3):280-90. doi: 10.1016/j.molcel.2009.06.010.
Degradation by the proteasome typically requires substrate ubiquitination. Two ubiquitin receptors exist in the proteasome, S5a/Rpn10 and Rpn13. Whereas Rpn13 has only one ubiquitin-binding surface, S5a binds ubiquitin with two independent ubiquitin-interacting motifs (UIMs). Here, we use nuclear magnetic resonance (NMR) and analytical ultracentrifugation to define at atomic level resolution how S5a binds K48-linked diubiquitin, in which K48 of one ubiquitin subunit (the "proximal" one) is covalently bonded to G76 of the other (the "distal" subunit). We demonstrate that S5a's UIMs bind the two subunits simultaneously with a preference for UIM2 binding to the proximal subunit while UIM1 binds to the distal one. In addition, NMR experiments reveal that Rpn13 and S5a bind K48-linked diubiquitin simultaneously with subunit specificity, and a model structure of S5a and Rpn13 bound to K48-linked polyubiquitin is provided. Altogether, our data demonstrate that S5a is highly adaptive and cooperative toward binding ubiquitin chains.
蛋白酶体介导的降解通常需要底物泛素化。蛋白酶体中存在两种泛素受体，即S5a/Rpn10和Rpn13。Rpn13只有一个泛素结合表面，而S5a通过两个独立的泛素相互作用基序（UIM）结合泛素。在这里，我们使用核磁共振（NMR）和分析型超速离心技术，以原子水平分辨率确定S5a如何结合K48连接的双泛素，其中一个泛素亚基（“近端”亚基）的K48与另一个（“远端”亚基）的G76共价结合。我们证明，S5a的UIM同时结合两个亚基，其中UIM2更倾向于结合近端亚基，而UIM1结合远端亚基。此外，NMR实验表明，Rpn13和S5a以亚基特异性同时结合K48连接的双泛素，并提供了S5a和Rpn13与K48连接的多聚泛素结合的模型结构。总之，我们的数据表明，S5a在结合泛素链方面具有高度的适应性和协同性。
