Ras S89D mutation induced allosteric changes that promoted its nucleotide exchange and signaling activation.

The small GTPase Ras is among the most frequently mutated genes and its mutations often drive oncogenesis across various cancers. While the role of NRas phosphorylation at S89 in the context of a Q61R mutation in melanoma genesis remains controversial, the impact of S89 phosphorylation on NRas function has not been fully elucidated. In this study, we employed the S89D phosphorylation-mimetic mutation and demonstrated that the S89D mutation alone activated all Ras isoforms by increasing the GTP-bound population, thereby promoting ERK phosphorylation and cell proliferation. The S89D mutant retained unaltered hydrolysis kinetics and GTP/GDP relative affinity but exhibited an accelerated intrinsic nucleotide exchange rate, due to impaired nucleotide binding. A 1.2 Å crystal structure of the S89D mutant revealed substantial local conformational changes, as well as alterations propagating to the nucleotide-binding pocket, providing a structural basis for the observed biochemical properties. Collectively, these findings established that the S89D mutation activated Ras by enhancing intrinsic nucleotide exchange, offering new insights into Ras allostery.