Fig 3. Ribbon depiction comparing dimethylated Lys-5 KRAS to that of WT KRAS obtained from three 200 ns MD simulations.

(A) Structural overlay of GDP-bound WT KRAS (blue, population ~83%) with GDP-bound dimethylated Lys-5 KRAS (black, population ~76%) and (B) GTP-bound WT (purple, population 86%) and GTP-bound dimethylated Lys-5 KRAS (green, population ~84%). Dimethylation of Lys-5 does not significantly affect packing interactions of Lys-5 with Y71 and T74 sidechains in the GDP-bound state or with T74 in the GTP-bound state. Average residue RMSF values and their standard errors obtained from three 200 ns MD trajectories comparing dimethylated Lys-5 (black) and WT (blue) KRAS for both (C) GDP- and (D) GTP-bound states. (E) Distances for key RAS/nucleotide interactions for unmodified and dimethylated RAS in both GDP- and GTP-bound states. Comparison shows similar distances, indicating proper nucleotide and magnesium association. These distances include the Cγ of Phe-28 to C4 of the guanine indole ring and Oγ of Ser-17 to Mg²⁺. All distances are calculated from three 200 ns MD trajectories for each system. Two distinct, key average distances between the two switch (Switch I and Switch II) regions are also compared, for wild type and Lys-5 dimethylated KRAS. Dimethylated Lys-5 KRAS does not significantly alter switch distances in either the GDP or GTP-bound states. Distances between O of Thr-35 and N of Gly-60 and between Cζ of Tyr-32 and Cδ of Gln-61 are shown. (F) Mutation of Lys-5 to Ala, Leu or Asn affects RAS activity. Flag-His tagged KRAS mutants were expressed in HEK293T cells, and their activation level analyzed by GST-RBD pull-down of RAS (see Materials and methods). Western blots with anti-Flag and anti-pan RAS antibodies reveal the relative activation levels of the KRAS mutants relative to endogenous RAS, respectively.