Structural Basis for the Specific Recognition of RhoA by the Dual GTPase-activating Protein ARAP3

Abstract

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 (ADPribosylation 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).

RACGAP1 variants in a sporadic case of CDA III implicate the dysfunction of centralspindlin as the basis of the disease

“…2A and S4A). We noticed that the region on CYK4 encompassing R385, L396, and P432 is located close to the GTPase residues that are distinct among RhoA, Cdc42, and Rac1 13,14 (Figs. 2A and S4B).…”

A rare congenital anemia is caused by mutations in the centralspindlin complex

“…Thus, we investigated whether these new CDAIII variants might affect the GAP activity of CYK4 towards RhoA, Rac1, and Cdc42. GTP hydrolysis by RhoA, Rac1, or Cdc42 was monitored by measuring the released free phosphate using an enzyme coupled photometric assay under a multiple turnover condition in which the GDP release is not rate-limiting (26,27). The GAP activity was determined as the increase of the GTPase rate in the presence of the CYK4 GAP domain (Fig.…”

Conformational Insights into the Control of CNF1 Toxin Activity by Peptidyl-Prolyl Isomerization: A Molecular Dynamics Perspective

“…The first criterion (i) was meant to provide a protein/peptide position agreeing with the deamidation reaction of Q63, whereas the second criterion (ii) was meant to obtain peptide conformations roughly fitting the folded structure of the G protein. The distance threshold for criterion (ii) was estimated from an analysis of the Cα-Cα distances between residues 60 and 72 in the RhoA structures corresponding to the following PDB entries: 1A2B [ 56 ], 1CC0 [ 57 ], 1CXZ [ 58 ], 1DPF [ 59 ], 1FTN [ 60 ], 1KMQ [ 61 ], 1LB1 [ 62 ], 1S1C [ 63 ], 1X86 [ 64 ], 1XCG [ 65 ], 2RGN [ 66 ], 3KZ1 [ 67 ], 3LW8 [ 68 ], 3LWN [ 68 ], 3LXR [ 68 ], 3MSX (to be published), 3T06 [ 69 ], 3TVD [ 70 ], 4D0N [ 71 ], 4F38 [ 72 ], 4XH9 [ 73 ], 4XOI [ 74 ], 4XSG [ 75 ], 4XSH [ 75 ], 5A0F [ 76 ], 5BWM [ 75 ], 5C2K (to be published), 5C4M (to be published), 5EZ6 (to be published), 5FR1 [ 77 ], 5FR2 [ 77 ], 5HPY [ 78 ], 5IRC [ 79 ], 5JCP [ 80 ], 5JHG (to be published), 5JHH (to be published), 6BC0 [ 81 ], 6BCA [ 68 ], and 6BCB [ 68 ]. This analysis revealed that the distances between the Cα atoms were in the range 9.4–10.8 Å.…”