Deconstruction of the Ras switching cycle through saturation mutagenesis

  1. Pradeep Bandaru
  2. Neel H Shah
  3. Moitrayee Bhattacharyya
  4. John P Barton
  5. Yasushi Kondo
  6. Joshua C Cofsky
  7. Christine L Gee
  8. Arup K Chakraborty
  9. Tanja Kortemme
  10. Rama Ranganathan  Is a corresponding author
  11. John Kuriyan  Is a corresponding author
  1. University of California, Berkeley, United States
  2. Howard Hughes Medical Institute, University of California, Berkeley, United States
  3. Ragon Institute of MGH, MIT and Harvard, United States
  4. Massachusetts Institute of Technology, United States
  5. University of California, San Francisco, United States
  6. University of Texas Southwestern Medical Center, United States
  7. Lawrence Berkeley National Laboratory, United States
10 figures and 2 additional files

Figures

Figure 1 with 5 supplements
The Ras switching cycle and the bacterial two-hybrid system.

(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 …

https://doi.org/10.7554/eLife.27810.002
Figure 1—figure supplement 1
Conservation of the GTPase domains of H-Ras.

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 …

https://doi.org/10.7554/eLife.27810.003
Figure 1—figure supplement 2
Optimization of the bacterial two-hybrid system.

(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 …

https://doi.org/10.7554/eLife.27810.004
Figure 1—figure supplement 3
Reproducibility of the bacterial two-hybrid system.

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.

https://doi.org/10.7554/eLife.27810.005
Figure 1—figure supplement 4
HPLC analysis of GMP-PNP loaded Ras.

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 …

https://doi.org/10.7554/eLife.27810.006
Figure 1—figure supplement 5
Raw ITC data for wild-type Ras.
https://doi.org/10.7554/eLife.27810.007
Figure 2 with 2 supplements
Mutational tolerance of Ras in the regulated-Ras experiment.

(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 …

https://doi.org/10.7554/eLife.27810.008
Figure 2—source data 1

Raw data for GAP- and GEF- stimulated nucleotide hydrolysis and exchange rates.

https://doi.org/10.7554/eLife.27810.009
Figure 2—figure supplement 1
Mutational sensitivity of structural elements involved in nucleotide and effector binding.

(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, …

https://doi.org/10.7554/eLife.27810.010
Figure 2—figure supplement 2
Validation of mutational effects through in vitro biochemical measurements of GAP- and GEF- stimulated nucleotide hydrolysis and exchange rates and a yeast growth assay.

(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 …

https://doi.org/10.7554/eLife.27810.011
Mutational tolerance of Ras in the attenuated-Ras experiment.

(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, …

https://doi.org/10.7554/eLife.27810.012
Figure 4 with 1 supplement
Mutational tolerance of Ras in the unregulated-Ras experiment.

(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 …

https://doi.org/10.7554/eLife.27810.013
Figure 4—figure supplement 1
Intrinsic nucleotide release rates.

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.

https://doi.org/10.7554/eLife.27810.014
Mutational tolerance of Ras in the Ras-G12V experiment.

(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 …

https://doi.org/10.7554/eLife.27810.015
Figure 6 with 2 supplements
Superposition of structures from molecular dynamics simulations of GTP-bound forms of Ras.

(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 …

https://doi.org/10.7554/eLife.27810.016
Figure 6—figure supplement 1
Superposition of structures from GTP-bound simulations of wild type, H27A, and Y157A mutants.

(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 …

https://doi.org/10.7554/eLife.27810.017
Figure 6—figure supplement 2
Flexibility in Ras measured by root mean squared fluctuations of Cα atoms.

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 …

https://doi.org/10.7554/eLife.27810.018
Figure 7 with 1 supplement
The impact of hotspot mutations on structural flexibility in Ras.

(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 …

https://doi.org/10.7554/eLife.27810.019
Figure 7—figure supplement 1
Residue contacts in Ras.

(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 …

https://doi.org/10.7554/eLife.27810.020
Figure 8 with 1 supplement
Sequence variation in Ras.

(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 …

https://doi.org/10.7554/eLife.27810.021
Figure 8—figure supplement 1
Effect of substitutions in human H-Ras that are present in S. rosetta Ras.

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.

https://doi.org/10.7554/eLife.27810.022
Figure 9 with 1 supplement
Interaction of Ras with the two Ras-binding sites of SOS.

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 …

https://doi.org/10.7554/eLife.27810.023
Figure 9—figure supplement 1
SOS-stimulated nucleotide exchange of S. rosetta and human Ras.

(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 …

https://doi.org/10.7554/eLife.27810.024
Figure 10 with 1 supplement
Comparison of the R and T states in Ras.

(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 …

https://doi.org/10.7554/eLife.27810.025
Figure 10—figure supplement 1
R and T states of Ras bound to SOS.

(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 …

https://doi.org/10.7554/eLife.27810.026

Additional files

Supplementary file 1

Raw fitness data from bacterial two-hybrid screen.

https://doi.org/10.7554/eLife.27810.027
Supplementary file 2

Crystallographic data collection and refinement statistics.

https://doi.org/10.7554/eLife.27810.028

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