Oncogenic RAS proteins, involved in similar to 30% of human tumors, are molecular
switches of various signal transduction pathways. Here we apply a new protocol for
the NMR study of KRAS in its (inactive) GDP- and (activated) GTP-bound form, allowing
a comprehensive analysis of the backbone dynamics of its WT-, G12C- and G12D variants.
We found that Tyr32 shows opposite mobility with respect to the backbone of its surroundings:
it is more flexible in the GDP-bound form while more rigid in GTP-complexes (especially
in WT- and G12D-GTP). Using the G12C/Y32F double mutant, we showed that the presence
of the hydroxyl group of Tyr32 has a marked effect on the G12C-KRAS-GTP system as
well. Molecular dynamics simulations indicate that Tyr32 is linked to the gamma-phosphate
of GTP in the activated states - an arrangement shown, using QM/MM calculations, to
support catalysis. Anchoring Tyr32 to the gamma-phosphate contributes to the capture
of the catalytic waters participating in the intrinsic hydrolysis of GTP and supports
a simultaneous triple proton transfer step (catalytic water -> assisting water ->
Tyr32 -> O1G of the gamma-phosphate) leading to straightforward product formation.
The coupled flip of negatively charged residues of switch I toward the inside of the
effector binding pocket potentiates ligand recognition, while positioning of Thr35
to enter the coordination sphere of the Mg(2+)widens the pocket. Position 12 mutations
do not disturb the capture of Tyr32 by the gamma-phosphate, but (partially) displace
Gln61, which opens up the catalytic pocket and destabilizes catalytic water molecules
thus impairing intrinsic hydrolysis.
