Bao Hongyu, Li Fudong, Wang Chongyuan, Wang Na, Jiang Yiyang, Tang Yajun, Wu Jihui, Shi Yunyu
From the Hefei National Laboratory for Physical Science at Microscale, Collaborative Innovation Center of Chemistry for Life Sciences and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027, China.
From the Hefei National Laboratory for Physical Science at Microscale, Collaborative Innovation Center of Chemistry for Life Sciences and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, 230027, China
J Biol Chem. 2016 Aug 5;291(32):16709-19. doi: 10.1074/jbc.M116.736140. Epub 2016 Jun 15.
ARAP3 (Arf-GAP with Rho-GAP domain, ANK repeat, and PH domain-containing protein 3) is unique for its dual specificity GAPs (GTPase-activating protein) activity for Arf6 (ADP-ribosylation factor 6) and RhoA (Ras homolog gene family member A) regulated by phosphatidylinositol 3,4,5-trisphosphate and a small GTPase Rap1-GTP and is involved in regulation of cell shape and adhesion. However, the molecular interface between the ARAP3-RhoGAP domain and RhoA is unknown, as is the substrates specificity of the RhoGAP domain. In this study, we solved the crystal structure of RhoA in complex with the RhoGAP domain of ARAP3. The structure of the complex presented a clear interface between the RhoGAP domain and RhoA. By analyzing the crystal structure and in combination with in vitro GTPase activity assays and isothermal titration calorimetry experiments, we identified the crucial residues affecting RhoGAP activity and substrates specificity among RhoA, Rac1 (Ras-related C3 botulinum toxin substrate 1), and Cdc42 (cell division control protein 42 homolog).
ARAP3（含RhoGAP结构域、锚蛋白重复序列和PH结构域的Arf-GAP蛋白3）具有独特的双特异性GTP酶激活蛋白（GAP）活性，可作用于由磷脂酰肌醇3,4,5-三磷酸以及小GTP酶Rap1-GTP调控的Arf6（ADP核糖基化因子6）和RhoA（Ras同源基因家族成员A），并参与细胞形态和黏附的调控。然而，ARAP3的RhoGAP结构域与RhoA之间的分子界面尚不清楚，RhoGAP结构域的底物特异性也未知。在本研究中，我们解析了与ARAP3的RhoGAP结构域结合的RhoA的晶体结构。该复合物的结构展示了RhoGAP结构域与RhoA之间清晰的界面。通过分析晶体结构，并结合体外GTP酶活性测定和等温滴定量热实验，我们确定了影响RhoGAP活性以及RhoA、Rac1（Ras相关的C3肉毒杆菌毒素底物1）和Cdc42（细胞分裂控制蛋白42同源物）之间底物特异性的关键残基。
