(A) Ras cycles between an active, GTP-bound state and an inactive, GDP-bound state. Ras•GTP binds to effector proteins, such as Raf kinase, which binds to Switch I. The intrinsic hydrolysis of GTP … see more
H-Ras displays a high level of sequence conservation in metazoans. Sequences of H-Ras orthologs that were most similar to human H-Ras were used in this analysis. Invertebrate Ras sequences, such as … see more
(A) Growth rate differences are observed for Ras variants that perturb Raf-RBD binding affinity in the presence of chloramphenicol. Cells rapidly grow to high density when no chloramphenicol is … see more
Correlation of relative enrichment values (ΔEx) for all four conditions in the presence and absence of regulation for two independent experiments. Reported ΔEx values are averaged over both experiments.
HPLC was used to confirm GMP-PNP loading of Ras variants according to Eberth and Ahmadian (Eberth and Ahmadian, 2009). A mix of nucleotides (GMP, GDP, GMP-PNP, and GTP) was first injected as a … see more
(A) The results of the regulated-Ras experiment are shown in the form of a 165 × 20 matrix. Each row of the matrix represents one of the 20 amino acids, and each column shows one of the residues of … see more
Raw data for GAP- and GEF- stimulated nucleotide hydrolysis and exchange rates.
(A) The Ras•GTP:Raf-RBD complex. Mutations to Ras that affect Raf-RBD binding result in a loss-of-function. Critical interactions at the interface (blue) involving residues Glu 31, Asp 33, Thr 35, … see more
(A) GAP-stimulated GTP hydrolysis rates for a panel of Ras mutants, as measured by a fluorescent sensor for inorganic phosphate release upon hydrolysis of GTP to GDP. Mutations in Switch I (residues … see more
(A) Relative enrichment values (ΔEx) are shown in matrix form as in Figure 2A, for the attenuated-Ras experiment. Mutations at residues known to be mutated in human cancer (e.g. Gly 12, Gly 13, Gln 61, … see more
(A) Relative enrichment values (ΔEx) are shown in matrix form as in Figure 2A, for the unregulated-Ras experiment. In this experiment, Ras is expressed without the GAP and the GEF, and hotspots of … see more
Intrinsic GDP release rates for a panel of Ras mutants, as measured by fluorescent nucleotide release. H27G, L120A, and Y157A mutants lead to increased GDP release rates.
(A) Relative enrichment values (ΔEx) are shown in matrix form as in Figure 2A, for the Ras-G12V experiment, in the absence of the GAP and the GEF. In this experiment, Ras shows a muted pattern of … see more
(A) Wild-type Ras•GTP. The diagrams show the superposition of 30 structures sampled evenly from two 300 ns trajectories. The diagram on the left shows a canonical view of Ras•GTP. The two other … see more
(A) Wild-type Ras. The diagrams show the superposition of 300 structures sampled evenly from two 300 ns trajectories. The diagram on the left shows a canonical view of Ras•GTP. The two other … see more
Fluctuations in Ras are measured by the root mean square (r.m.s.) fluctuations of Cα atoms in Ras over the course of the 600 ns molecular dynamics trajectories. The r.m.s. fluctuations for wild-type … see more
(A) Sustained sidechain-sidechain contacts in the simulation of wild-type Ras•GTP. We used the network analysis tool of Vishveshwara and co-workers (Bhattacharyya et al., 2013) to analyze the … see more
(A) The crystal structure of wild-type Ras•GTP with the sidechains of His 94 and Tyr 137 shown as sticks. (B–C) Shown here are 30 structures sampled from two 600 ns simulations, one for wild-type … see more
(A) An evolutionary tree from an alignment of 72 extant Ras sequences from invertebrates (blue) and vertebrates (orange). The hypothetical ancestral sequence at the base of the tree is highlighted … see more
The substitutions in human H-Ras that are present in the S. rosetta Ras sequence are compared to the mutational data from the unregulated-Ras experiment.
Structure of the Ras:SOS complex (PDB code: 1NVV). Two molecules of H-Ras are bound to the allosteric and active site of SOS. The allosteric site of SOS is bound by Ras•GTP, whereas the active site … see more
(A) Human SOS stimulates nucleotide exchange for S. rosetta Ras•GDP in the presence of excess GDP in solution, however, no increase in exchange rate is observed for S. rosetta Ras•GDP in the … see more
(A) Variable Regions 2 and 3 highlighted on the structure of Ras•GTP. Variable Region 2 comprises helix α3 and the preceding loop, and Variable Region 3 comprises helix α4 and the preceding loop … see more
(A) Ras in the T state bound to the active site of SOS (left). Many sidechain contacts are present between the Switch II helix (red) and SOS (grey), including the sidechain of Tyr 71. Ras in the R … see more
Raw fitness data from bacterial two-hybrid screen.
Crystallographic data collection and refinement statistics.