Department of Early Discovery Biochemistry, Genentech Inc., South San Francisco, CA 94080, USA; Department of Discovery Chemistry, Genentech Inc., South San Francisco, CA 94080, USA.
Department of Structural Biology, Genentech Inc., South San Francisco, CA 94080, USA.
Structure. 2018 Jan 2;26(1):72-84.e7. doi: 10.1016/j.str.2017.11.010. Epub 2017 Dec 14.
Ubiquitin-specific protease 7 (USP7) deubiquitinase activity is controlled by a number of regulatory factors, including stimulation by intramolecular accessory domains. Alone, the USP7 catalytic domain (USP7cd) shows limited activity and apo USP7cd crystal structures reveal a disrupted catalytic triad. By contrast, ubiquitin-conjugated USP7cd structures demonstrate the canonical cysteine protease active-site geometry; however, the structural features of the USP7cd that stabilize the inactive conformation and the mechanism of transition between inactive and active states remain unclear. Here we use comparative structural analyses, molecular dynamics simulations, and in silico sequence re-engineering via directed sampling by RosettaDesign to identify key molecular determinants of USP7cd activation and successfully engineer USP7cd for improved activity. Full kinetic analysis and multiple X-ray crystal structures of our designs indicate that electrostatic interactions in the distal "switching loop" region and local packing in the hydrophobic core mediate subtle but significant conformational changes that modulate USP7cd activation.
泛素特异性蛋白酶 7（USP7）去泛素化酶活性受到许多调节因子的控制，包括分子内辅助结构域的刺激。单独的 USP7 催化结构域（USP7cd）显示出有限的活性，而无辅因子的 USP7cd 晶体结构显示出破坏的催化三联体。相比之下，连接有泛素的 USP7cd 结构表现出典型的半胱氨酸蛋白酶活性位点几何形状；然而，稳定非活性构象的 USP7cd 的结构特征以及在非活性和活性状态之间转变的机制仍不清楚。在这里，我们使用比较结构分析、分子动力学模拟以及通过 RosettaDesign 的定向采样进行的计算序列重新设计，来确定 USP7cd 激活的关键分子决定因素，并成功设计出用于提高活性的 USP7cd。我们的设计的完整动力学分析和多个 X 射线晶体结构表明，远端“开关环”区域的静电相互作用和疏水区的局部堆积介导了微妙但显著的构象变化，从而调节 USP7cd 的激活。
