KRAS switch loop movements play a crucial role in regulating RAS signaling, and alteration of these sensitive dynamics is a principal mechanism through which disease-associated RAS mutations lead to aberrant RAS activation. Prior studies suggest that despite a high degree of sequence similarity, the switches in KRAS are more dynamic than those in HRAS. We determined X-ray crystal structures of the rare tumorigenic KRAS mutants KRASD33E, in switch 1 (SW1), and KRASA59G, in switch 2 (SW2), bound to GDP and found these adopt nearly identical, open SW1 conformations as well as altered SW2 conformations. KRASA59G bound to a GTP analogue crystallizes in the same conformation. This open conformation is consistent with the inactive "state 1" previously observed for HRAS bound to GTP. For KRASA59G, switch rearrangements may be regulated by increased flexibility in the 57DXXGQ61 motif at codon 59. However, loss of interactions between side chains at codons 33 and 35 in the SW1 33DPT35 motif drives changes for KRASD33E. The 33DPT35 motif is conserved for multiple members of the RAS subfamily but is not found in RAB, RHO, ARF, or Gα families, suggesting that dynamics mediated by this motif may be important for determining the selectivity of RAS-effector interactions. Biochemically, the consequence of altered switch dynamics is the same, showing impaired interaction with the guanine exchange factor SOS and loss of GAP-dependent GTPase activity. However, interactions with the RBD of RAF are preserved. Overall, these observations add to a body of evidence suggesting that HRAS and KRAS show meaningful differences in functionality stemming from differential protein dynamics independent of the hypervariable region.
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