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

Abstract

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.

Fig. 1. Analysis of wild-type and mutant RHOA biochemical properties.

a Nucleotide dissociation assays for RHOA^WT (teal), RHOA^F30L (cyan), RHOA^A161P (orange), RHOA^A161V (purple) with RHOA-BODIPY-FL-GDP: GTPγS ratio of 1:10 in the presence of Mg²⁺. Data represents mean ± SD with n = 4 biological repeats. b Statistical analysis (unpaired t-test) of dissociation rates obtained from fitting data in a. Data represents mean ± SD. c Pull-down assays with MBP-LARG-DH/PH to detect binding of purified wild-type and mutant RHOA proteins preloaded with GDP or GMPPNP or in the presence of EDTA. The pull-down has been done with or without supplemented nucleotides. The pull-down has been repeated three times with similar results. d Measurement of RHOA intrinsic or GST-p190-GAP stimulated GTPase activity. GTPase activity of wild-type and mutant RHOA at different concentrations was measured with or without 2 μM GST-P190-GAP. Data represents mean ± SD with n = 4–6 biological repeats. e Statistical analysis (unpaired t-test) of EC50s from fitting data in d. Data represents mean ± SD. RHOA^T37A is significantly different from the others and the P value is not shown (P < 0.00001). Source data are provided as a Source Data file.

Fig. 2. ³¹P NMR spectra of GDP-, GMPPNP-, and GTPγS-bound RHOA^WT and mutants.

a Comparison of ³¹P NMR spectra of GDP-bound RHOA^WT (0.84 mM), RHOA^F30L (0.71 mM), RHOA^A161P (0.40 mM), and RHOA^A161V (0.63 mM) at 275 K. Both RHOA^A161V and RHOA^F30L showed an additional state at the α-phosphate that possibly has an open conformation around the α-phosphate. This state was barely detectable in RHOA^A161P. b Comparison of ³¹P NMR spectra of GMPPNP-bound RHOA^WT (0.67 mM), RHOA^F30L (0.62 mM), RHOA^A161P (0.45 mM), RHOA^A161V (0.44 mM), RHOA^T37A (0.51 mM) at 275 K. c Comparison of ³¹P NMR spectra of GTPγS-bound RHOA^WT (0.60 mM), RHOA^F30L (0.63 mM), RHOA^A161P (0.56 mM), RHOA^A161V (0.67 mM), and RHOA^T37A (0.7 mM) at 275 K. At the γ-phosphate, RHOA^A161P was very similar to RHOA^WT. In contrast, RHOA^A161V and RHOA^F30L displayed two inactive states, state 1 as in RHOA^WT and state 1’ as in RHOA^T37A. To better understand the nature of the states, ³¹P NMR spectra of RHOA^A161P (d), RHOA^A161V (e), and RHOA^F30L (f) in complex with GST-RHOTEKIN-RBD were collected at 275 K. RHOA^WT titrations with GST-RHOTEKIN-RBD and GST control have been published previously. The molar ratio of GST-RHOTEKIN-RBD/RHOA increases from 0, 0.5, 1, to 2, starting from ~0.5 mM RHOA proteins. All spectra were aligned with the inorganic phosphate Pi peak.

Fig. 3. Structural analysis and MD simulation of RHOA^A161P-GDP and RHOA^A161V-GDP.

a Overall fold of GDP-bound RHOA^A161P and RHOA^A161V. The switch regions are colored orange (RHOA^A161P) or purple (RHOA^A161V), encompassing residues 28–40 and 60–78, respectively. b Two conserved water molecules mediate a hydrogen-bond network between the nucleotide guanine base and RHOA, as exemplified in RHOA^A161V-GDP. Electron densities around the GDP, the two water molecules, residue 161, and other surrounding residues are contoured at 1σ (wheat) from the 2Fo-Fc map. c These two water molecules are missing in RHOA^A161P-GDP. d Backbone RMSF plot for RHOA-GDP systems. e The distances of important residues and GDP. Y34, T37, and G62 distances were calculated using C_α of the residues and β-phosphate. Source data are provided as a Source Data file.

Fig. 4. GDP-bound mutant RHOA^A161V and RHOA^A161P interact with effectors.

a Pull-down assay with GST-RHOTEKIN-RBD and GST-ROCK2-RBD to detect the binding of purified wild-type and mutant RHOA proteins preloaded with GDP or GMPPNP. The pull-down has been repeated five times with similar results. b Microscale thermophoresis analysis of the direct binding between GDP- (black) or GMPPNP-bound (red) RHOA proteins and RHOTEKIN-RBD. ΔFnorm [‰] = 10*(Fnorm(bound) − Fnorm(unbound)). Data represents mean ± SD with n = 8 biological repeats for RHOA^WT-GMPPNP and n = 3 for all other samples. K_D values obtained from the binding curves are listed and shown in μM. Source data are provided as a Source Data file.

Fig. 5. RHOA^A161V-GDP showed a population of active conformation in the ¹H-¹⁵N HSQC spectra.

a Plot of peak intensities against residue number for non-overlapping assigned peaks. Intensities were normalized to the average peak intensity from assigned, non-overlapping peaks in the RHOA^WT-GDP spectrum. The domain structure of truncated RHOA (residues 1–181) is aligned at the top of the plot and the red asterisk (*) is the mutational site. b Plot of CSPs for assigned peaks against residue number. For V38 and S160, the “active” peak (most similar to RHOA^WT-GMPPNP) in the RHOA^A161V-GDP spectrum was used to calculate the CSPs. c ¹H-¹⁵N HSQC spectra of GDP- and GMPPNP-bound RHOA^WT and RHOA^A161V indicated that RHOA^A161V-GDP had two populations, as exemplified by residue V38 (top panel) and S160 (bottom panel). d Average intensities of ¹H-¹⁵N HSQC spectra, normalized to the corresponding RHOA samples prior to RHOTEKIN addition, were plotted against ratios of titrated RHOTEKIN-RBD to RHOA. The error bars are the standard deviation of the averaged intensities. e Titration of 2X RHOTEKIN-RBD to RHOA^A161V-GDP indicated that for the doubled peaks for V38 (left panel) and S160 (right panel). Black: apoprotein; Red: with 2X RHOTEKIN-RBD. f Titration curve of peak intensities for V38. The error bars are the same as in d. The “active” peak (solid black curve) in RHOA^A161V-GDP had a greater decrease in intensity upon binding than the other (dotted black curve). In d and f, the spectrum of each sample at each condition was collected once. Source data are provided as a Source Data file.