Structural Basis for the Specific Recognition of RhoA by the Dual GTPase-activating Protein ARAP3.
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).
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.
Hongyu
H
Bao
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.
Fudong
0000-0002-0986-0491
F
Li
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.
Chongyuan
C
Wang
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.
Na
N
Wang
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.
Yiyang
Y
Jiang
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.
Yajun
Y
Tang
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 wujihui@ustc.edu.cn.
Jihui
0000-0002-4339-0518
J
Wu
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 yyshi@ustc.edu.cn.
Yunyu
0000-0002-1348-4759
Y
Shi
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains; D000089804:Shelterin Complex; D013329:Structure-Activity Relationship; D034501:Telomere-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000737:chemistry* / Q000235:genetics
D000089982:ADP-Ribosylation Factor 6; C446512:ARAP3 protein, human; D048868:Adaptor Proteins, Signal Transducing; D020690:GTPase-Activating Proteins; D018129:Phosphatidylinositol Phosphates; D000089804:Shelterin Complex; C440809:TERF2IP protein, human; D034501:Telomere-Binding Proteins; C060974:phosphatidylinositol 3,4,5-triphosphate; C066737:RHOA protein, human; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D020742:rhoA GTP-Binding Protein
ARAP3; GTPase activity assay; GTPase-activating protein (GAP); Ras homolog gene family, member A (RhoA); Rho (Rho GTPase); RhoGAP; complex; crystal structure; substrate specificity