A saturation-mutagenesis analysis of the interplay between stability and activation in Ras
- California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, United States
- Howard Hughes Medical Institute, University of California, Berkeley, United States
- Department of Chemistry, University of California, Berkeley, United States
- Department of Molecular and Cell Biology, University of California, Berkeley, United States
- Department of Chemistry, Columbia University, United States
- Biophysics Graduate Group, University of California, Berkeley, United States
Figures
Figure 1 with 1 supplement
Ras G-domain, the switching cycle, and schematics of the two selection assays.
(A) The three principal sites of cancer mutations in Ras, referred to as the three cancer hotspots – Gly 12, Gly 13, and Gln 61 – are shown in the K-Ras structure. The C-terminal helix extension is …
Figure 1—figure supplement 1
Analysis of the distribution of Ras mutations in COSMIC v94.
(A) Counts for each Ras variant that appears in the Catalogue of Somatic Mutations in Cancer (COSMIC) v94 database (Tate et al., 2019). The counts for each isoform have been aggregated and grouped …
Figure 2 with 2 supplements
Mutational tolerance of Ras in mammalian Ba/F3 cells and the bacterial Ras+GAP experiment.
(A) The fitness data from saturation-mutagenesis experiments are shown in the form of a matrix, where each row of the matrix represents one of the twenty natural amino acids, and each column …
Figure 2—figure supplement 1
Mammalian Ba/F3 cell screen replicates and growth curves of wild-type H-Ras versus the G12V mutant.
(A) Scatter plot of the enrichment scores of the two biological replicates of the mammalian Ba/F3 screen. (B) Growth curves of mammalian Ba/F3 cells transduced with wild-type H-Ras or H-Ras G12V. …
Figure 2—figure supplement 2
Mutational tolerance of Ras in the bacterial Ras+GAP+GEF experiment.
(A) Relative enrichment values () are shown in matrix form for the following experiments: K-Ras2−165+GAP+GEF and K-Ras2−173+GAP+GEF. The enrichment values of each experiment displayed are the mean …
Figure 3 with 1 supplement
Mutational tolerance of Ras long and short constructs in the unregulated bacterial experiment.
(A) H-Ras2-166, H-Ras2-180, K-Ras2-165, and K-Ras2-173 in the unregulated experiment. The relative enrichment values () shown are the mean of three, four, two, and four biological replicates, …
Figure 3—figure supplement 1
ROC analysis between the mammalian Ba/F3 cell experiment and the unregulated bacterial H-Ras and K-Ras experiments.
Scores are lower than for the Ras+GAP experiments.
Figure 4
Thermodynamic stability measurements.
(A) The shorter H-Ras1-166 construct has an unfolding free-energy change (ΔGunf) of 22.2 ± 1.6 kJ⋅mol-1, and the longer H-Ras1-173 construct has a ΔGunf of 29.9 ± 1.4 kJ⋅mol-1 when measured by …
Figure 5 with 3 supplements
Hydrogen to deuterium exchange by NMR for activating H-Ras mutants.
(A) Schematic of expected backbone hydrogen to deuterium exchange (HDX) over time when lyophilized protein is resolubilized in D2O. (B) Structure displaying the four H-Ras mutants analyzed by HDX …
Figure 5—figure supplement 1
Thermodynamic stability measurements of L120A and Q99A.
Pulse proteolysis measurements of H-Ras1-166 L120A and Q99A variants, as well as new replicates of the wild-type construct. The values of the unfolding free-energy change (ΔGunf) are 14.0 ± 0.3 …
Figure 5—figure supplement 2
Relative change in hydrogen to deuterium exchange for each mutant.
Relative exchange (kobs,mutant/kobs,WT) plotted over residue number. A map of the secondary structural elements is aligned at the bottom. The structural overlay of the same data can be seen in Figure…
Figure 5—figure supplement 3
Wild-type H-Ras exhibits EX1 behavior at the N and C terminus.
HDX was measured by mass spectrometry. Reactions were quenched at different time points from time 0 to 24 hr and the protein was digested. Peptide m/z (residue numbers labeled at the top of each …
Figure 6 with 2 supplements
Prediction of the cancer mutations by the mammalian Ba/F3 cell and Ras+GAP experiments.
(A) Counts per residue of Ras variants that appear in the COSMIC v94 database and pass the five-count cutoff (Tate et al., 2019). There are twenty-eight residues where at least one mutation is …
Figure 6—figure supplement 1
Prediction of the cancer mutations by the bacterial experiments.
This figure shows the ROC plots for the experiments not shown in Figure 6B. The Ras+GAP datasets have higher ROC AUC scores than the Ras+GAP+GEF or the unregulated conditions.
Figure 6—figure supplement 2
Prediction of the cancer mutations using the cBioPortal dataset as true positives.
(A) Scatter plot between the COSMIC counts and the cBioPortal counts per variant. (B) The ROC analysis shown in Figure 6 and Figure 6—figure supplement 1 was repeated using the cBioPortal counts as …
Figure 7 with 4 supplements
Analysis of the phenotype of activating hotspots in the screens (A) Comparison of the mutational sensitivity profiles for long and short H-Ras+GAP datasets.
The pairwise difference (epistasis) is shown averaged over amino acids at each position (). Figure 7—figure supplement 4 compiles the activating sites where there is epistasis. (B) Comparison of …
Figure 7—figure supplement 1
Structures of C-Raf-RBD, PI3K, p120GAP, and SOScat in complex with H-Ras, and SOScat-stimulated GTP release measurements of selected mutants.
Switch II is involved in binding to SOS, p120GAP, and effectors such as PI3K, but not C-Raf-RBD. (A) C-Raf-RBD•H-Ras complex, (B) PI3K•H-Ras complex, (C) p120GAP•H-Ras complex, and (D) H-Ras•SOScat•H…
Figure 7—figure supplement 2
Mutational tolerance of K-Ras2-173 with a Q61L background mutation.
(A) Relative enrichment values () are shown in matrix form for the K-Ras2-173 Q61L (‘Q61L’) construct in the absence of regulators and Ras+GAP conditions. The enrichment values of each experiment …
Figure 7—figure supplement 3
Location of top-activating sites in the mammalian Ba/F3 cell experiment and structure of H-Ras, Rap1B, and Rab29.
(A) Structure of H-Ras with the top-nine sites of gain-of-function mutations in the H-Ras1-188 in mammalian Ba/F3 cells. PDB ID: 5P21 (Pai et al., 1990). (B) The G-domain is conserved among the …
Figure 7—figure supplement 4
Sites of H-Ras where mutations have a different phenotype in the mammalian Ba/F3 cell and H-Ras+GAP experiments.
The column ‘In cancer databases’ lists if mutations at that residue are found in cancer, regardless of the number of counts. The columns ‘COSMIC 5+ counts’ and ‘cBioPortal 5+ counts’ say whether a …
Figure 8
Signaling activity versus thermodynamic stability.
A conceptual relationship between the thermodynamic stability and signaling activity of Ras variants is shown. A decrease in stability can increase signaling activity by enhancing …
Author response image 1
Tables
Author response table 1
| Construct | Wild-type | L120A | Y157Q | H27G | Q99A |
|---|---|---|---|---|---|
| Number of peaks | 127 | 109 | 104 | 82 (after 1 hour of deuterium exchange) | 113 |
