KRAS K104 modification affects the KRASG12D-GEF interaction and mediates cell growth and motility

Abstract

Mutant RAS genes play an important role in regulating tumors through lysine residue 104 to impair GEF-induced nucleotide exchange, but the regulatory role of KRAS K104 modification on the KRASG12D mutant remains unclear. Therefore, we simulated the acetylation site on the KRASG12D three-dimensional protein structure, including KRASG12D, KRASG12D/K104A and KRASG12D/K104Q, and determined their trajectories and binding free energy with GEF. KRASG12D/K104Q induced structural changes in the α2- and α3-helices, promoted KRAS instability and hampered GEF binding (ΔΔG = 6.14 kJ/mol). We found decreased binding to the Raf1 RBD by KRASG12D/K104Q and reduced cell growth, invasion and migration. Based on whole-genome cDNA microarray analysis, KRASG12D/K104Q decreased expression of NPIPA2, DUSP1 and IL6 in lung and ovarian cancer cells. This study reports computational and experimental analyses of Lys104 of KRASG12D and GEF, and the findings provide a target for exploration for future treatment.

Allosteric regulation of switch-II controls K-Ras oncogenicity

“…We substituted lysine for glutamine to mimic constitutive acetylation of K104, however, it is not entirely clear how well this mutation really phenocopies actual acetylation. For example, consistent with our prior and current work, multiple groups demonstrated that the K104Q mutation interrupts interaction with GEF leading to reduction of GEF-induced nucleotide exchange, but other study found that acetylation of K104 did not [13, 31, 32]. Our modeled structure of K-Ras with acetylated K104 suggests that acetylation of K104 should reduce GEF-induced nucleotide exchange in K-Ras.…”

“…Nucleotide binding controls K-Ras functional activity by influencing its conformational state, primarily at switch I (SWI; residues [30][31][32][33][34][35][36][37][38] and switch II (SWII: residues 60-76). Although both switch regions interact with GEF, GAP, and downstream effector proteins, SWI primarily facilitates GTP hydrolysis through GAP binding and SWII plays a significant role in binding to GEF to promote nucleotide exchange.…”