Withers-Ward E S, Mueller T D, Chen I S, Feigon J
Department of Microbiology, University of California at Los Angeles, Los Angeles, California 90095, USA.
Biochemistry. 2000 Nov 21;39(46):14103-12. doi: 10.1021/bi0017071.
The DNA repair protein HHR23A is a highly conserved protein that functions in nucleotide excision repair. HHR23A contains two ubiquitin associated domains (UBA) that are conserved in a number of proteins with diverse functions involved in ubiquitination, UV excision repair, and signaling pathways via protein kinases. The cellular binding partners of UBA domains remain unclear; however, we previously found that the HHR23A UBA(2) domain interacts specifically with the HIV-1 Vpr protein. Analysis of the low resolution solution structure of HHR23A UBA(2) revealed a hydrophobic loop region of the UBA(2) domain that we predicted was the interface for protein/protein interactions. Here we present results of in vitro binding studies that demonstrate the requirement of this hydrophobic loop region for interaction with human immunodeficiency virus (HIV-1) Vpr. A single point mutation of the Pro at residue 333 to a Glu totally abolishes the binding of HIV-1 Vpr to UBA(2). High resolution NMR structures of the binding deficient UBA(2) mutant P333E as well as of the wild-type UBA(2) domain were determined to compare the effect of this mutation on the structure. Small but significant differences are observed only locally at the site of the mutation. The biochemical and structural analysis confirms the function of the HHR23A UBA(2) GFP-loop as the protein/protein interacting domain.
DNA修复蛋白HHR23A是一种高度保守的蛋白，在核苷酸切除修复中发挥作用。HHR23A包含两个泛素相关结构域（UBA），在许多具有不同功能的蛋白中保守，这些功能涉及泛素化、紫外线切除修复以及通过蛋白激酶的信号通路。UBA结构域的细胞结合伴侣仍不清楚；然而，我们之前发现HHR23A UBA(2)结构域与HIV-1 Vpr蛋白特异性相互作用。对HHR23A UBA(2)低分辨率溶液结构的分析揭示了UBA(2)结构域的一个疏水环区域，我们预测该区域是蛋白/蛋白相互作用的界面。在此，我们展示了体外结合研究的结果，这些结果证明了该疏水环区域对于与人类免疫缺陷病毒（HIV-1）Vpr相互作用的必要性。残基333处的脯氨酸单点突变为谷氨酸完全消除了HIV-1 Vpr与UBA(2)的结合。测定了结合缺陷型UBA(2)突变体P333E以及野生型UBA(2)结构域的高分辨率NMR结构，以比较该突变对结构的影响。仅在突变位点局部观察到微小但显著的差异。生化和结构分析证实了HHR23A UBA(2) GFP环作为蛋白/蛋白相互作用结构域的功能。
