Biochemical and biophysical characterization of the RAS family small GTPase protein DiRAS3.

DiRAS3, also called ARHI, is a RAS (sub)family small GTPase protein that shares 50-60% sequence identity with H-, K-, and N-RAS, with substitutions in key conserved G-box motifs and a unique 34 amino acid extension at its N-terminus. Unlike the RAS proto-oncogenes, DiRAS3 exhibits tumor suppressor properties. DiRAS3 function has been studied through genetics and cell biology, but there has been a lack of understanding of the biochemical and biophysical properties of the protein, likely due to its instability and poor solubility. To overcome this solubility issue, we engineered a DiRAS3 variant (C75S/C80S), which significantly improved soluble protein expression in E. coli. Recombinant DiRAS3 was purified by Ni-NTA and size exclusion chromatography (SEC). Concentration dependence of the SEC chromatogram indicated that DiRAS3 exists in monomer-dimer equilibrium. We then produced truncations of the N-terminal (ΔN) and both (ΔNC) extensions to the GTPase domain. Unlike full-length DiRAS3, the SEC profiles showed that ΔNC is monomeric while ΔN was monomeric with aggregation, suggesting that the N and/or C-terminal tail(s) contribute to dimerization and aggregation. The 1H-15N HSQC NMR spectrum of ΔNC construct displayed well-dispersed peaks similar to spectra of other GTPase domains, which enabled us to demonstrate that DiRAS3 has a GTPase domain that can bind GDP and GTP. Taken together, we conclude that, despite the substitutions in the G-box motifs, DiRAS3 can switch between nucleotide-bound states and that the N- and C-terminal extensions interact transiently with the GTPase domain in intra- and inter-molecular fashions, mediating weak multimerization of this unique small GTPase. Copyright © 2023 Elsevier Inc. All rights reserved.

Citation

Takashi Saitoh, Ha-Neul Kim, Riku Narita, Ibuki Ohtsuka, Weiyu Mo, Ki-Young Lee, Masahiro Enomoto, Geneviève M C Gasmi-Seabrook, Christopher B Marshall, Mitsuhiko Ikura. Biochemical and biophysical characterization of the RAS family small GTPase protein DiRAS3. Protein expression and purification. 2023 Dec;212:106361

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PMID: 37652393

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