The pathogenic G361A variant of CRAF, associated with increased intrinsic kinase activity in Noonan syndrome, remains poorly understood in terms of its molecular and structural impact on kinase activity. To elucidate the mechanistic implications of the glycine to alanine substitution at residue 361 in CRAF, we employed molecular dynamics simulations. Our findings reveal that this mutation predominantly affects the ATP binding pocket and critical intermolecular interactions within the active cleft that favors the phosphate transfer reaction. Notably, our data highlight significant alterations in key interactions involving Lys470/Asp486 and ATP.Mg2+ in CRAFG361A that are absent in wild-type CRAF. Additionally, we identified a novel interaction mode between Lys431 and γ-phosphate in wild-type CRAF, a residue evolutionarily conserved in CRAFs but not in related kinases such as BRAF, ARAF, and KSR1/2. Furthermore, observed shifts in the αC-helix and G-loop relative to the wild-type correlate with an enlarged ATP-binding cavity in the mutant, reflecting structural adaptations due to these mutations. Overall, these structural insights underscore the elevated intrinsic kinase activity of the CRAFG361A variant and provide crucial mechanistic details that could inform the development of specific inhibitors targeting this variant.

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