Duda David M, Borg Laura A, Scott Daniel C, Hunt Harold W, Hammel Michal, Schulman Brenda A
Howard Hughes Medical Institute, St Jude Children's Research Hospital, Memphis, TN 38105, USA.
Cell. 2008 Sep 19;134(6):995-1006. doi: 10.1016/j.cell.2008.07.022.
Cullin-RING ligases (CRLs) comprise the largest ubiquitin E3 subclass, in which a central cullin subunit links a substrate-binding adaptor with an E2-binding RING. Covalent attachment of the ubiquitin-like protein NEDD8 to a conserved C-terminal domain (ctd) lysine stimulates CRL ubiquitination activity and prevents binding of the inhibitor CAND1. Here we report striking conformational rearrangements in the crystal structure of NEDD8Cul5(ctd)-Rbx1 and SAXS analysis of NEDD8Cul1(ctd)-Rbx1 relative to their unmodified counterparts. In NEDD8ylated CRL structures, the cullin WHB and Rbx1 RING subdomains are dramatically reoriented, eliminating a CAND1-binding site and imparting multiple potential catalytic geometries to an associated E2. Biochemical analyses indicate that the structural malleability is important for both CRL NEDD8ylation and subsequent ubiquitination activities. Thus, our results point to a conformational control of CRL activity, with ligation of NEDD8 shifting equilibria to disfavor inactive CAND1-bound closed architectures, and favor dynamic, open forms that promote polyubiquitination.
Cullin-RING连接酶（CRLs）构成了最大的泛素E3亚类，其中央cullin亚基将底物结合衔接蛋白与E2结合RING连接起来。泛素样蛋白NEDD8与保守的C末端结构域（ctd）赖氨酸的共价连接刺激CRL泛素化活性，并阻止抑制剂CAND1的结合。在此，我们报告了NEDD8Cul5（ctd）-Rbx1晶体结构中的显著构象重排以及相对于未修饰对应物的NEDD8Cul1（ctd）-Rbx1的小角X射线散射（SAXS）分析。在NEDD8化的CRL结构中，cullin的WHB和Rbx1的RING亚结构域发生了显著重排，消除了一个CAND1结合位点，并为相关的E2赋予了多种潜在的催化几何结构。生化分析表明，结构可塑性对CRL的NEDD8化和随后的泛素化活性都很重要。因此，我们的结果表明CRL活性存在构象控制，NEDD8的连接使平衡发生移动，不利于无活性的CAND1结合的封闭结构，而有利于促进多聚泛素化的动态开放形式。
