Tumor-derived RHOA mutants interact with effectors in the GDP-bound state

RHOA mutations are found at diverse residues in various cancer types, implying mutation- and cell-specific mechanisms of tumorigenesis. Here, we focus on the underlying mechanisms of two gain-of-function RHOA mutations, A161P and A161V, identified in adult T-cell leukemia/lymphoma. We find that RHOA^A161P and RHOA^A161V are both fast-cycling mutants with increased guanine nucleotide dissociation/association rates compared with RHOA^WT and show reduced GTP-hydrolysis activity. Crystal structures reveal an altered nucleotide association in RHOA^A161P and an open nucleotide pocket in RHOA^A161V. Both mutations perturb the dynamic properties of RHOA switch regions and shift the conformational landscape important for RHOA activity, as shown by ³¹P NMR and molecular dynamics simulations. Interestingly, RHOA^A161P and RHOA^A161V can interact with effectors in the GDP-bound state. ¹H-¹⁵N HSQC NMR spectra support the existence of an active population in RHOA^A161V-GDP. The distinct interaction mechanisms resulting from the mutations likely favor an RHOA^WT-like "ON" conformation, endowing GDP-bound state effector binding activity.