Isoform-Specific Destabilization of the Active Site Reveals a Molecular Mechanism of Intrinsic Activation of KRas G13D

Abstract

Ras GTPases are mutated at codons 12, 13, and 61, with different frequencies in KRas, HRas, and NRas and in a cancer-specific manner. The G13D mutant appears in 25% of KRas-driven colorectal cancers, while observed only rarely in HRas or NRas. Structures of Ras G13D in the three isoforms show an open active site, with adjustments to the D13 backbone torsion angles and with disconnected switch regions. KRas G13D has unique features that destabilize the nucleotide-binding pocket. In KRas G13D bound to GDP, A59 is placed in the Mg²⁺ binding site, as in the HRas-SOS complex. Structure and biochemistry are consistent with an intermediate level of KRas G13D bound to GTP, relative to wild-type and KRas G12D, observed in genetically engineered mouse models. The results explain in part the elevated frequency of the G13D mutant in KRas over the other isoforms of Ras.

Keywords: KRas G12D; KRas G13D mouse model; KRas G13D structures; Ras GTPases; Ras conformational states; activation cycle; colorectal cancer; nucleotide exchange mutant; oncogene.

Figure 1.. Ras G13D in the GTP-Bound State Favors an Open Active Site

(A) The G-domain of Ras with effector (light gray) and allosteric lobes (light blue). Wild-type KRas-GppCH₂p with switch I in state 1 (PDB: 5UK9, magenta). HRas-GppNHp in state 2 can have switch II in the disordered T-state (PDB: 2RGE, gray) or the R-state (PDB: 3K8Y, green). (B) Ras G13D structures in state 1: H13GNP (orange), Ha13GNP (bright orange), Hb13GNP (light orange), Hc13GNP (yellow), Ka13GNP (cyan), Kb13GNP (light teal), and N13GNP (purple), with wild-type KRas as in (A). (C) Ramachandran plot showing the ϕ,ψ dihedral angles for P loop residues 10–14 in wild type (gray), G12D (blue), and G13D (orange) for the Ras structures shown in Table S1. Generally allowed regions are contoured in blue and those allowed for glycine are in green (empirical data compiled by Lovell et al., 2003). Yellow-filled circles enclose ϕ,ψ angles for residues other than D13 and red circles enclose ϕ,ψ angles for D13 in the G13D structures. (D) Ras G13D disrupts the interaction between switches I and II at the beginning of the β2 and β3 sheets. The β2 and β3 H-bonding interactions are shown for wild-type HRas as black dashed lines and for the G13D mutant as yellow dashed lines. Wild-type HRas is in green and the Ha13GNP model is in bright orange. See also Figure S1 and Table S1.

Figure 2.. KRas G13D Structures in the GTP-Bound and GDP-Bound Forms

(A) H95 in KRas G13D (Ka13GNP, brown), nestled between Q99 and Y96, affects residues in the nucleotide-binding pocket relative to wild-type KRas (PDB: 5UK9, magenta). H-bonds are shown as black dashed lines for wild-type KRas and as yellow dashed lines for Ka13GNP. (B) View of the active site with electron density (blue wire mesh) for the Kb13GNP structure (yellow), with disordered F28 and K147. (C) The K13GDP model (bright brown) shows D13 coordinated to a Na⁺ ion (purple) and linked to switch II and helix 3 residue H95 through water-mediated H-bonding interactions (yellow dashed lines). (D) The N-terminal end of switch II in K13GDP (bright brown, yellow dashed lines) with A59 in the Mg²⁺ binding site, superimposed on switch II of wild-type HRas bound to SOS (PDB: 1BKD, green sticks, black dashed lines). Electron density contoured at the 1 σ level is shown in (B) and (D) (blue wired mesh). See also Figure S2.

Figure 3.. G13D Favors State 1 More Prominently Than Wild Type in Both KRas and HRas

(A and B) ¹H-NMR spectra for (A) wild-type KRas and KRas G13D and (B) wild-type HRas and HRas G13D bound to GppNHp, showing the temperature dependence for each peak. The spectra for the wild-type proteins (black) were previously discussed, along with peak contributions (Parker et al., 2018), also described in Method Details. (C) Superposition of the Hc13GNP model (gold) with that of the Ras/Raf-RBD complex (PDB: 4G0N; HRas green, Raf-RBD magenta). (D) D13 of Hc13GNP (gold) clashes with the position of Y32 in wild-type HRas in state 2 (PDB: 3K8Y, green). D13 helps coordinate a Na⁺ ion (purple) in the active site. Water molecules are shown in the black sphere for wild-type HRas and in red for Hc13GNP, with orange dashed lines representing H-bonding interactions in the mutant. See also Figures S3, S4, S5, and S6.

Figure 4.. KRas G13D Has a Distinct Phenotype from that of Wild-Type KRas and KRas G12D in the Murine Colonic Epithelium

(A) H&E staining of formalin-fixed and paraffin-embedded colon Swiss rolls (5 μm) from 8- to 12-week-old mice. Scale bar, 200 μm. (B) Crypt height measurements across the entire length of the colon from H&E stains in (A). Measurements were taken every 5 crypts, and the curves are composites of measurements from 7 wild type (Fabpl-Cre), 7 G13D, and 5 G12D. (C) Quantification of data in (B). (D) Affinity precipitation of Ras-GTP using Raf-RBD. Each pull-down used 500 μg colon lysate from 8- to 12-week-old mice, while total Ras blot was done with 50 μg lysate. Right, quantification of band intensities from 3 wild-type, 4 G13D, and 3 G12D pull-down experiments. (E) Representative western blot of wild-type, G13D, and G12D colon lysate for phosphorylated-to-total protein from Erk1/2, pan-Akt, and its two phosphorylation sites T308 and S473. Right, quantitation of western blot bands from 11 wild type, 12 G13D, and 12 G12D biological replicates for Erk and pAkt S473, and 10 wild-type, 11 G13D, and 10 G12D biological replicates for pAkt T308; ***p < 0.0001, 1-way ANOVA (Kruskal-Wallis). Error bars of quantitated western blot data represent SEMs (±SEMs). (F) Response of murine colonic organoids of various KRas alleles to 6 days of Akt (MK2206) and Erk (SCH772984) treatment. Data show luminescence averages from three biological replicates per genotype, and curves were fit with nonlinear regression. Error bars of luminescence data ± SEMs.

Figure 5.. Mechanism for Cycling between GDP- and GTP-Bound Forms of Ras G13D

Models in cyan depict each step of the catalytic cycle, with the preceding step shown in black. Residues are labeled in the central panel (3), and these residues are shown in their respective conformations in each panel. (1) PDB: 4TQA, (2) Hb13GNP, (3) K13GDP, (4) Ka13GNP, and (5) Hc13GNP. Na⁺ is shown in purple and Mg²⁺ is shown in green. Note that A59 is disordered in (4) and therefore not present in the model shown in cyan.