RNF12 is a widely expressed ubiquitin E3 ligase that is required for X-chromosome inactivation, regulation of LIM-domain containing transcription factors, and TGF-β signaling. A RING domain at the C terminus of RNF12 is important for its E3 ligase activity, and mutations in the RING domain are associated with X-linked intellectual disability. Here we have characterized ubiquitin transfer by RNF12, and show that the RING domain can bind to, and is active with, ubiquitin conjugating enzymes (E2s) that produce degradative ubiquitin chains. We report the crystal structures of RNF12 in complex with two of these E2 enzymes, as well as with an E2~Ub conjugate in a closed conformation. These structures form a basis for understanding the deleterious effect of a number of disease causing mutations. Comparison of the RNF12 structure with other monomeric RINGs suggests that a loop prior to the core RING domain has a conserved and essential role in stabilization of the active conformation of the bound E2~Ub conjugate. Together these findings provide a framework for better understanding substrate ubiquitylation by RNF12 and the impact of disease causing mutations.

RNF12是广泛表达的泛素E3连接酶，是X染色体失活，调节包含LIM结构域的转录因子和TGF-β信号传导所必需的。RNF12 C末端的RING域对其E3连接酶活性很重要，并且RING域中的突变与X连锁的智力障碍有关。在这里，我们已经表征了RNF12介导的遍在蛋白转移，并显示RING域可以与产生降解遍在蛋白链的遍在蛋白缀合酶（E2s）结合并具有活性。我们报告了RNF12的晶体结构，其中有两个与这些E2酶以及一个封闭构象的E2〜Ub共轭物复合。这些结构构成了了解许多引起突变的疾病的有害作用的基础。RNF12结构与其他单体RING的比较表明，在核心RING域之前的环在稳定结合的E2〜Ub共轭物的活性构象方面具有保守和必不可少的作用。这些发现共同为更好地理解RNF12的底物泛素化以及疾病引起突变的影响提供了框架。