Oncogenic G12D mutation alters local conformations and dynamics of K-Ras

Abstract

K-Ras is the most frequently mutated oncoprotein in human cancers, and G12D is its most prevalent mutation. To understand how G12D mutation impacts K-Ras function, we need to understand how it alters the regulation of its dynamics. Here, we present local changes in K-Ras structure, conformation and dynamics upon G12D mutation, from long-timescale Molecular Dynamics simulations of active (GTP-bound) and inactive (GDP-bound) forms of wild-type and mutant K-Ras, with an integrated investigation of atomistic-level changes, local conformational shifts and correlated residue motions. Our results reveal that the local changes in K-Ras are specific to bound nucleotide (GTP or GDP), and we provide a structural basis for this. Specifically, we show that G12D mutation causes a shift in the population of local conformational states of K-Ras, especially in Switch-II (SII) and α3-helix regions, in favor of a conformation that is associated with a catalytically impaired state through structural changes; it also causes SII motions to anti-correlate with other regions. This detailed picture of G12D mutation effects on the local dynamic characteristics of both active and inactive protein helps enhance our understanding of local K-Ras dynamics, and can inform studies on the development of direct inhibitors towards the treatment of K-Ras^G12D-driven cancers.

Figure 1

K-Ras switch mechanism and 3D structure. (A) Cartoon representation of K-Ras switch mechanism between GTP-bound (active) to GDP-bound (inactive) forms and the GTP hydrolysis reaction. (B) Cartoon representation of K-Ras protein consisting of secondary structures. Blue: β-sheets, green: α-helices, pink: P-loop, light pink: SI region, purple: SII region. (C) Zoomed-in visualization of the P-loop. Grey: Wild-type, pink: G12D mutant. Residues G12 and D12 are in stick representation. (D) G12D mutation site (red), protein-protein interaction site residues for its regulator and downstream effector proteins (green) and schematic representation of K-Ras sequence (residues 1–169).

Figure 2

Conformational changes in K-Ras upon G12D mutation. (A) The differences in average pairwise distances ($\mathrm{\Delta }{\overline{R}}_{ij}$) where K-Ras^WT is the reference state. Red dots (positive $\mathrm{\Delta }{\overline{R}}_{ij}$ values) show that pairs move further apart and blue dots (negative $\mathrm{\Delta }\overline{R}ij$ values) show that pairs move closer in K-Ras^G12D. (B) The average of all $\mathrm{\Delta }{\overline{R}}_{ij}$ values for each residue, $\mathrm{\Delta }\overline{R}i$. The initial state is K-Ras^WT and the final state is K-Ras^G12D. The residues which move away from their neighbors have positive values and dominate the mutant protein; residues that move close to their neighbors have negative values.

Figure 3

Distribution of distances between residue pairs in SII-α3 region. Distance distributions of Cα of residue pairs in K-Ras^WT-GTP (black) and K-Ras^G12D-GTP (red) (A) Q61-D92, (B) E62-H95, (C) Y64-H95; in K-Ras^WT-GDP (grey) and K-Ras^G12D-GDP (pink) (D) Q61-D92, (E) E62-H95, (F) Y64-H95.

Figure 4

Distribution of distances between residue pairs in the P loop-SII region. Distance distributions between Cαs of residue pairs in K-Ras^WT-GTP (black) and K-Ras^G12D-GTP (red) (A) A11-Q61, (B) G12D-Q61, (C) G12D-Q61; in K-Ras^WT-GDP (grey) and K-Ras^G12D-GDP (pink) (D) A11-Q61, (E) G12D-Q61, (F) G12D-Q61.

Figure 5

Distance distributions of residue pairs which get closer after G12D mutation in K-Ras-GTP in K-Ras^WT (black) and K-Ras^G12D (red). Distance distribution of Cαs of residue pairs in K-Ras^WT-GTP (black) and K-Ras^G12D-GTP (red) (A) L23-V152, (B) L23-F156; in K-Ras^WT-GDP (grey) and K-Ras^G12D-GDP (pink) (C) L23-V152, (D) L23-F156.

Figure 6

Dynamic changes in K-Ras upon G12D mutation. (A) RMSF values of K-Ras^WT-GTP (black) and K-Ras^G12D-GTP (red) residues. (B) RMSF values of K-Ras^WT-GDP (grey) and K-Ras^G12D-GDP (pink) residues.

Figure 7

The maps of correlation coefficients from the motions of correlated residues in (A) K-Ras^WT-GTP, (B) K-Ras^G12D-GTP, (C) K-Ras^WT-GDP and (D) K-Ras^G12D-GDP. Red dots show positive correlations and blue dots show negative correlations.