The small GTPase MRAS is a broken switch
Nature Communications volume 16, Article number: 647 (2025 ) Cite this article
Intense research on founding members of the RAS superfamily has defined our understanding of these critical signalling proteins, leading to the premise that small GTPases function as molecular switches dependent on differential nucleotide loading. The closest homologs of H/K/NRAS are the three-member RRAS family, and interest in the MRAS GTPase as a regulator of MAPK activity has recently intensified. We show here that MRAS does not function as a classical switch and is unable to exchange GDP-to-GTP in solution or when tethered to a lipid bilayer. The exchange defect is unaffected by inclusion of the GEF SOS1 and is conserved in a distal ortholog from nematodes. Synthetic activating mutations widely used to study the function of MRAS in a presumed GTP-loaded state do not increase exchange, but instead drive effector binding due to sampling of an activated conformation in the GDP-loaded state. This includes nucleation of the SHOC2-PP1Cα holophosphatase complex. Acquisition of NMR spectra from isotopically labeled MRAS in live cells validated the GTPase remains fully GDP-loaded, even a supposed activated mutant. These data show that RAS GTPases, including those most similar to KRAS, have disparate biochemical activities and challenge current dogma on MRAS, suggesting previous data may need reinterpretation.
Principal gatekeepers of signal transduction are small GTPase proteins, which typically function as ‘molecular switches’ by exchanging between two structural conformations. In the active, GTP-bound state they bind effector proteins that initiate signaling cascades directing growth, differentiation, survival, and motility. A conformational change elicited by GDP in the active site prevents effector binding. Three GTPase homologs are fundamental regulators of normal development and drivers of human cancers. Point mutations lock HRAS, KRAS and NRAS (hereon H/K/NRAS or classical RAS) in an activated state, driving constitutive proliferation1. Their impact on human health has made H/K/NRAS foremost targets of cancer therapeutics and a major research focus for four decades, meaning this subset of proteins has come to define our understanding of small GTPase function. Cancer-causing point mutations locking H/K/NRAS in a GTP-bound state have been widely used to enable study of the large ~160 member RAS superfamily, which includes GTPases of the RAS, RHO, ARF, RAB and RAN subgroups2. This is despite a notable lack of biochemical data exploring the function of most family members, though the nucleotide switch is believed a fundamental property of these signaling enzymes. The few exceptions include atypical RHO GTPases (RND class3 and RHOH4) that bind GTP but have no enzymatic activity, and RHOBTB proteins that do not bind nucleotides5,6. These proteins are now classified as pseudoGTPases7. RGK proteins of the RAS subfamily (REM1, REM2, GEM and RRAD) may constitute another class, as they do not undergo conformational change with nucleotide cycling8,9, and some ARF GTPases do not bind nucleotides or have significantly higher affinity for GTP10.
