Dharadhar Shreya, Clerici Marcello, van Dijk Willem J, Fish Alexander, Sixma Titia K
Division of Biochemistry and CGC.nl, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Division of Biochemistry and CGC.nl, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands; Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland.
J Struct Biol. 2016 Dec;196(3):437-447. doi: 10.1016/j.jsb.2016.09.011. Epub 2016 Sep 17.
Regulation of deubiquitinating enzyme (DUB) activity is an essential step for proper function of cellular ubiquitin signals. UAF1 is a WD40 repeat protein, which binds and activates three important DUBs, USP1, USP12 and USP46. Here, we report the crystal structure of the USP12-Ub/UAF1 complex at a resolution of 2.8Å and of UAF1 at 2.3Å. In the complex we find two potential sites for UAF1 binding, analogous to what was seen in a USP46/UAF1 complex. In line with these observed dual binding states, we show here that USP12/UAF1 complex has 1:2 stoichiometry in solution, with a two-step binding at 4nM and 325nM respectively. Mutagenesis studies show that the fingers sub-domain of USP12 interacts with UAF1 to form the high affinity interface. Our activation studies confirm that the high affinity binding is important for activation while the second UAF1 binding does not affect activation. Nevertheless, we show that this two step binding is conserved in the well-studied USP12 paralog, USP1. Our results highlight the interfaces essential for regulation of USP12 activity and show a conserved second binding of UAF1 which could be important for regulatory functions independent of USP12 activity.
去泛素化酶（DUB）活性的调节是细胞泛素信号正常功能的关键步骤。UAF1是一种WD40重复蛋白，它结合并激活三种重要的DUB，即USP1、USP12和USP46。在此，我们报告了分辨率为2.8Å的USP12-Ub/UAF1复合物以及分辨率为2.3Å的UAF1的晶体结构。在复合物中，我们发现了两个UAF1结合的潜在位点，类似于在USP46/UAF1复合物中观察到的情况。与这些观察到的双重结合状态一致，我们在此表明USP12/UAF复合物在溶液中的化学计量比为1:2，分别在4nM和325nM时存在两步结合。诱变研究表明，USP12的指状亚结构域与UAF1相互作用形成高亲和力界面。我们的激活研究证实，高亲和力结合对激活很重要，而UAF1的第二次结合不影响激活。然而，我们表明这种两步结合在所研究的USP12旁系同源物USP1中是保守的。我们的结果突出了调节USP12活性所必需的界面，并显示了UAF1保守的第二次结合，这可能对独立于USP12活性的调节功能很重要。
