K-Ras Populates Conformational States Differently from Its Isoform H-Ras and Oncogenic Mutant K-RasG12D  Jillian A. Parker, Alicia Y. Volmar, Spiro Pavlopoulos, Carla Mattos  Structure  Volume 26, Issue 6, Pages 810-820.e4 (June 2018) DOI: 10.1016/j.str.2018.03.018 Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 1 Conserved Motifs in Ras GTPase The P loop (10GAGGVGKS17) is shown in yellow, 57DTAG60, as well as catalytic residue Q61, are in dark green, the 116NKCD119 motif is in blue, and the 143ETSAK147 motif is in magenta. Switch I residues mentioned in the text are in red and so are the oxygen atoms of water molecules. The Mg2+ ion is in light green. The figure is a composite of two H-Ras structures. The protein, nucleotide, Mg2+ and water molecules heavy atoms are as in the coordinates with PDB: 3K8Y, with deuterium atoms added at locations on GppNHp and Mg2+ coordinating water molecules as observed in the neutron crystal structure with PDB: 4RSG. The deuterium atoms are shown in light gray and labeled with D. Structure 2018 26, 810-820.e4DOI: (10.1016/j.str.2018.03.018) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 2 WT K-Ras-GppCH2p Is Conformationally Distinct from the WT H-Ras-GppNHp Isoform (A) WT K-Ras-GppCH2p (green) aligned with WT H-Ras-GppNHp (gray, PDB: 3K8Y) highlights differences in the switch I and switch II regions. (B) WT K-Ras-GppCH2p exhibits a state 1 conformation with T35 away from the active site, where a third water molecule replaces the hydroxyl side chain to coordinate the Mg2+ (light green). Waters are shown as red spheres. The electron density for the 2Fo−Fc electron density map is shown in blue and contoured to 1σ. Structure 2018 26, 810-820.e4DOI: (10.1016/j.str.2018.03.018) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 3 1H NMR Spectra of WT K-Ras and its Mutants Bound to GDP, GppNHp, and WT K-Ras-GppNHp in Complex with Raf-RBD at 275 K Peaks labeled D and T denote a difference in chemical shift seen for the downfield resonance near 13 ppm in K-Ras-GDP versus K-Ras proteins bound to GppNHp, respectively. Important resonances in the 10.0–11.0 ppm range are labeled as A, B, X, and Z. See Figure S1 for 31P NMR spectrum of K-Ras-GppNHp. For assignments of peaks T and X see Figures S2–S4. For an educated guess on the proton associated with peak Z see Figure S5. For the spectra of K-Ras bound to GppCH2p and GTPγ-S see Figure S6. Structure 2018 26, 810-820.e4DOI: (10.1016/j.str.2018.03.018) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 4 1H NMR Spectra of WT H-Ras and its Mutants Bound to GppNHp at 275 K Peaks labeled D and T denote a difference in chemical shift seen for the downfield resonance near 13 ppm in H-Ras-GDP versus H-Ras proteins bound to a GppNHp, respectively. Important resonances in the 10.0–11.0 ppm range are labeled as A, B, X, and Z. See Figure S1 for 31P NMR spectrum of H-Ras-GppNHp. Structure 2018 26, 810-820.e4DOI: (10.1016/j.str.2018.03.018) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 5 Comparison of WT K-Ras and Oncogenic K-RasG12D (A) Inactive K-RasG12D-GDP (cyan, PDB: 4EPR) exhibits a unique conformation of Q61 in switch II that is not observed in WT K-Ras-GDP, where Q61 is disordered (wheat, molecule A from PDB: 5UK9). (B) In the K-RasG12D-GppNHp structure (purple, PDB: 4DSN) where switch 1 is “closed” over the active site, the Y32 and D12 side chains are coordinating the catalytically relevant bridging water molecule, while Q61 accepts an H bond from D12. The nucleophilic water molecule is also present in this structure. Both active site water molecules are absent in state 1 WT K-Ras-GppCH2p (green, molecule B from PDB: 5UK9). The state 2 switch I conformation of K-RasG12D-GppNHp is nearly identical to that in WT H-Ras-GppNHp (gray, PDB: 3K8Y). Structure 2018 26, 810-820.e4DOI: (10.1016/j.str.2018.03.018) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 6 Temperature Dependence of 1H Chemical Shifts in the H-Ras Proteins (A–C) 1H spectra were collected for (A) WT H-Ras-GppNHp, (B) H-RasT35S-GppNHp, and (C) H-RasY32A-GppNHp at 275, 280, 285, 288, and 295 K. Structure 2018 26, 810-820.e4DOI: (10.1016/j.str.2018.03.018) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 7 Temperature Dependence of 1H Chemical Shifts in the K-Ras Proteins (A–D) 1H spectra were collected for (A) WT K-Ras-GppNHp, (B) K-RasT35S-GppNHp, (C) K-Ras-GppNHp in complex with Raf-RBD, and (D) K-RasG12D-GppNHp at 275, 280, 285, 288, and 295 K. Structure 2018 26, 810-820.e4DOI: (10.1016/j.str.2018.03.018) Copyright © 2018 Elsevier Ltd Terms and Conditions