Search Menu
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information
Search Scholarly Publications

Title: Deconstruction of the Ras switching cycle through saturation mutagenesis

Journal Article · · eLife
ORCiD logo [1]; ORCiD logo [2];  [2]; ORCiD logo [3];  [2];  [2]; ORCiD logo [2];  [4];  [5];  [6]; ORCiD logo [7]
  1. Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Univ. of California, Berkeley, CA (United States). California Inst. for Quantitative Biosciences; Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; DOE/OSTI
  2. Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Univ. of California, Berkeley, CA (United States). California Inst. for Quantitative Biosciences; Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.
  3. Massachusetts Inst. of Technology (MIT) and Harvard Univ., Cambridge, MA (United States). Ragon Inst. of MGH; Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Dept. of Chemical Engineering; Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Dept. of Physics; Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Inst. for Medical Engineering and Science
  4. Massachusetts Inst. of Technology (MIT) and Harvard Univ., Cambridge, MA (United States). Ragon Inst. of MGH; Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Dept. of Chemical Engineering; Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Dept. of Physics; Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States). Inst. for Medical Engineering and Science; Dept. of Chemistry; Dept. of Biological Engineering
  5. Univ. of California, San Francisco, CA (United States). California Inst. for Quantitative Biomedical Research. Dept. of Bioengineering and Therapeutic Sciences
  6. Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Pharmacology; Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Green Center for Systems Biology; Univ. of Texas Southwestern Medical Center, Dallas, TX (United States). Dept. of Biophysics
  7. Univ. of California, Berkeley, CA (United States). Dept. of Molecular and Cell Biology; Univ. of California, Berkeley, CA (United States). California Inst. for Quantitative Biosciences; Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Inst.; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Molecular Biophysics and Integrated Bioimaging Division
+ Show Author Affiliations

Ras proteins are highly conserved signaling molecules that exhibit regulated, nucleotide-dependent switching between active and inactive states. The high conservation of Ras requires mechanistic explanation, especially given the general mutational tolerance of proteins. Here, we use deep mutational scanning, biochemical analysis and molecular simulations to understand constraints on Ras sequence. Ras exhibits global sensitivity to mutation when regulated by a GTPase activating protein and a nucleotide exchange factor. Removing the regulators shifts the distribution of mutational effects to be largely neutral, and reveals hotspots of activating mutations in residues that restrain Ras dynamics and promote the inactive state. Evolutionary analysis, combined with structural and mutational data, argue that Ras has co-evolved with its regulators in the vertebrate lineage. Overall, our results show that sequence conservation in Ras depends strongly on the biochemical network in which it operates, providing a framework for understanding the origin of global selection pressures on proteins.

Research Organization:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Organization:
USDOE Office of Science (SC)
Grant/Contract Number:
AC02-05CH11231
OSTI ID:
1628873
Journal Information:
eLife, Journal Name: eLife Vol. 6; ISSN 2050-084X
Publisher:
eLife Sciences Publications, Ltd.Copyright Statement
Country of Publication:
United States
Language:
English

References (159)

Ras activation by SOS: Allosteric regulation by altered fluctuation dynamics journal July 2014
Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. journal August 1990
The GTPase superfamily: conserved structure and molecular mechanism journal January 1991
Mapping the Nucleotide and Isoform-Dependent Structural and Dynamical Features of Ras Proteins journal June 2008
A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile journal May 2014
Collaboration gets the most out of software journal September 2013
The Guanine Nucleotide-Binding Switch in Three Dimensions journal November 2001
Robustness–epistasis link shapes the fitness landscape of a randomly drifting protein journal November 2006
How different amino acid sequences determine similar protein structures: The structure and evolutionary dynamics of the globins journal January 1980
Activation of prokaryotic transcription through arbitrary protein–protein contacts journal April 1997
Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. journal August 1990
Differences in the Regulation of K-Ras and H-Ras Isoforms by Monoubiquitination journal November 2013
The Genomic and Cellular Foundations of Animal Origins journal November 2013
Residue specific contributions to stability and activity inferred from saturation mutagenesis and deep sequencing journal February 2014
Origins of Allostery and Evolvability in Proteins: A Case Study journal July 2016
Residue specific contributions to stability and activity inferred from saturation mutagenesis and deep sequencing journal February 2014
Accurate whole human genome sequencing using reversible terminator chemistry journal November 2008
Integrated RAS signaling defined by parallel NMR detection of effectors and regulators journal January 2014
An Improved General Amino Acid Replacement Matrix journal April 2008
Selecting optimal second-line tyrosine kinase inhibitor therapy for chronic myeloid leukemia patients after imatinib failure: does the BCR-ABL mutation status really matter? journal December 2009
A Network Representation of Protein Structures: Implications for Protein Stability journal December 2005
Critical Binding and Regulatory Interactions between Ras and Raf Occur through a Small, Stable N-Terminal Domain of Raf and Specific Ras Effector Residues journal August 1994
Activation of prokaryotic transcription through arbitrary protein–protein contacts journal April 1997
A novel switch region regulates H-ras membrane orientation and signal output journal February 2008
Solving structures of protein complexes by molecular replacement with Phaser journal December 2006
The Species Specificity of Myoglobin journal November 1954
Mutational effects and the evolution of new protein functions journal August 2010
Ras Conformational Switching: Simulating Nucleotide-Dependent Conformational Transitions with Accelerated Molecular Dynamics journal March 2009
‘Latent drivers’ expand the cancer mutational landscape journal June 2015
The Pfam protein families database: towards a more sustainable future journal December 2015
Improved side-chain torsion potentials for the Amber ff99SB protein force field journal January 2010
Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins journal February 1990
RAS isoforms and mutations in cancer at a glance journal March 2016
PHENIX: a comprehensive Python-based system for macromolecular structure solution journal January 2010
Protein Structure and Function: Looking through the Network of Side-Chain Interactions journal November 2015
Scaling and assessment of data quality journal December 2005
Structural Evidence for Feedback Activation by Ras·GTP of the Ras-Specific Nucleotide Exchange Factor SOS journal March 2003
The COSMIC (Catalogue of Somatic Mutations in Cancer) database and website journal June 2004
PHENIX: a comprehensive Python-based system for macromolecular structure solution. text January 2010
Robustness–epistasis link shapes the fitness landscape of a randomly drifting protein journal November 2006
PASTA: Ultra-Large Multiple Sequence Alignment for Nucleotide and Amino-Acid Sequences journal May 2015
Pseudo-merohedral twinning in monoclinic crystals of wild-type human brain neuroglobin journal March 2009
Characterization of the Second Ion-Binding Site in the G Domain of H-Ras journal November 2012
Membrane-dependent signal integration by the Ras activator Son of sevenless journal May 2008
Entwicklung von Methoden zur Strukturvorhersage von Proteinkomplexen und zur Untersuchung von Proteinzuständen mit Hochdruckkernspinresonanzspektroskopie text January 2016
Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli journal June 2009
Mating pair stabilization mediates bacterial conjugation species specificity journal June 2022
The Ras protein superfamily: Evolutionary tree and role of conserved amino acids journal January 2012
PASTA: Ultra-Large Multiple Sequence Alignment for Nucleotide and Amino-Acid Sequences journal May 2015
Evolvability as a Function of Purifying Selection in TEM-1 β-Lactamase journal February 2015
Protein Structure and Function: Looking through the Network of Side-Chain Interactions journal November 2015
The small GTPases K-Ras, N-Ras, and H-Ras have distinct biochemical properties determined by allosteric effects journal June 2017
The GTPase superfamily: conserved structure and molecular mechanism journal January 1991
Mechanisms of Autoinhibition and STI-571/Imatinib Resistance Revealed by Mutagenesis of BCR-ABL journal March 2003
Thermodynamics of Ras/Effector and Cdc42/Effector Interactions Probed by Isothermal Titration Calorimetry journal April 2001
REFMAC 5 for the refinement of macromolecular crystal structures journal March 2011
Recognizing and Defining True Ras Binding Domains I: Biochemical Analysis journal May 2005
Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues. journal August 1994
Scaling and assessment of data quality journal December 2005
Selecting optimal second-line tyrosine kinase inhibitor therapy for chronic myeloid leukemia patients after imatinib failure: does the BCR-ABL mutation status really matter? journal December 2009
FastML: a web server for probabilistic reconstruction of ancestral sequences journal May 2012
Structure of Hæmoglobin: A Three-Dimensional Fourier Synthesis at 5.5-Å. Resolution, Obtained by X-Ray Analysis journal February 1960
Structural Analysis of Autoinhibition in the Ras Activator Son of Sevenless journal October 2004
Structure of Hæmoglobin: A Three-Dimensional Fourier Synthesis at 5.5-Å. Resolution, Obtained by X-Ray Analysis journal February 1960
Structural Analysis of Autoinhibition in the Ras Activator Son of Sevenless journal October 2004
Improved side-chain torsion potentials for the Amber ff99SB protein force field journal January 2010
The RAF proteins take centre stage journal November 2004
Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia journal August 2002
The Guanine Nucleotide-Binding Switch in Three Dimensions journal November 2001
Allosteric modulation of Ras positions Q61 for a direct role in catalysis journal March 2010
A Comprehensive Survey of Ras Mutations in Cancer journal May 2012
Pseudo-merohedral twinning in monoclinic crystals of wild-type human brain neuroglobin journal March 2009
Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae. journal December 1986
Analyses of the Effects of All Ubiquitin Point Mutants on Yeast Growth Rate journal April 2013
Alternative packing arrangements in the hydrophobic core of λrepresser journal May 1989
A Network Representation of Protein Structures: Implications for Protein Stability journal December 2005
Dynamic properties of the Ras switch I region and its importance for binding to effectors journal April 2001
Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site journal May 2015
Alternative packing arrangements in the hydrophobic core of λrepresser journal May 1989
RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies journal January 2014
Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site journal May 2015
K-Ras protein as a drug target journal March 2016
Differences in the Regulation of K-Ras and H-Ras Isoforms by Monoubiquitination journal November 2013
Deep mutational scanning: a new style of protein science journal July 2014
Solving structures of protein complexes by molecular replacement with Phaser journal December 2006
Resistance in the land of molecular cancer therapeutics journal August 2002
Characterization of the Second Ion-Binding Site in the G Domain of H-Ras journal November 2012
XDS journal January 2010
The spatial architecture of protein function and adaptation journal October 2012
Premetazoan genome evolution and the regulation of cell differentiation in the choanoflagellate Salpingoeca rosetta journal January 2013
Overview of the CCP 4 suite and current developments journal March 2011
GTP-Dependent K-Ras Dimerization journal July 2015
Pervasive degeneracy and epistasis in a protein-protein interface journal February 2015
The Pfam protein families database: towards a more sustainable future journal December 2015
Evolvability as a Function of Purifying Selection in TEM-1 β-Lactamase journal February 2015
Mapping the Nucleotide and Isoform-Dependent Structural and Dynamical Features of Ras Proteins journal June 2008
Allosteric Effects of the Oncogenic RasQ61L Mutant on Raf-RBD journal March 2015
Dynamic properties of the Ras switch I region and its importance for binding to effectors journal April 2001
Space-group and origin ambiguity in macromolecular structures with pseudo-symmetry and its treatment with the program Zanuda journal August 2014
Improved Binding of Raf to Ras·GDP Is Correlated with Biological Activity journal September 2009
Mechanisms of Autoinhibition and STI-571/Imatinib Resistance Revealed by Mutagenesis of BCR-ABL journal March 2003
Viewing Protein Fitness Landscapes Through a Next-Gen Lens journal October 2014
A study of communication pathways in methionyl- tRNA synthetase by molecular dynamics simulations and structure network analysis journal September 2007
Direct, Real-Time Measurement of Rapid Inorganic Phosphate Release Using a Novel Fluorescent Probe and Its Application to Actomyosin Subfragment 1 ATPase journal July 1994
A five-site model for liquid water and the reproduction of the density anomaly by rigid, nonpolarizable potential functions journal May 2000
Integrated RAS signaling defined by parallel NMR detection of effectors and regulators journal January 2014
The small GTPases K-Ras, N-Ras, and H-Ras have distinct biochemical properties determined by allosteric effects journal June 2017
Improved Binding of Raf to Ras·GDP Is Correlated with Biological Activity journal September 2009
Coot model-building tools for molecular graphics journal November 2004
Allosteric modulation of Ras positions Q61 for a direct role in catalysis journal March 2010
REFMAC 5 for the refinement of macromolecular crystal structures journal March 2011
The spatial architecture of protein function and adaptation journal October 2012
A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions journal June 2000
A study of communication pathways in methionyl- tRNA synthetase by molecular dynamics simulations and structure network analysis journal September 2007
A fast SHAKE algorithm to solve distance constraint equations for small molecules in molecular dynamics simulations journal January 2001
Space-group and origin ambiguity in macromolecular structures with pseudo-symmetry and its treatment with the program Zanuda journal August 2014
How ras-related proteins talk to their effectors journal December 1996
XDS journal January 2010
GTP-Dependent K-Ras Dimerization journal July 2015
Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae. journal December 1986
An automated approach to network features of protein structure ensembles: An Automated Approach to Network Features of Protein Structure Ensembles journal September 2013
Recognizing and Defining True Ras Binding Domains I: Biochemical Analysis journal May 2005
The RAF proteins take centre stage journal November 2004
Thermodynamics of Ras/Effector and Cdc42/Effector Interactions Probed by Isothermal Titration Calorimetry journal April 2001
Genomic and Biological Characterization of Exon 4 KRAS Mutations in Human Cancer journal June 2010
The Ras-RasGAP Complex: Structural Basis for GTPase Activation and Its Loss in Oncogenic Ras Mutants journal July 1997
Analyses of the Effects of All Ubiquitin Point Mutants on Yeast Growth Rate journal April 2013
A universal cloning method based on yeast homologous recombination that is simple, efficient, and versatile journal May 2014
Coot model-building tools for molecular graphics journal November 2004
Ras Conformational Switching: Simulating Nucleotide-Dependent Conformational Transitions with Accelerated Molecular Dynamics journal March 2009
Ras activation by SOS: Allosteric regulation by altered fluctuation dynamics journal July 2014
A fast SHAKE algorithm to solve distance constraint equations for small molecules in molecular dynamics simulations journal January 2001
Digital Signaling and Hysteresis Characterize Ras Activation in Lymphoid Cells journal January 2009
Origins of Allostery and Evolvability in Proteins: A Case Study journal July 2016
How different amino acid sequences determine similar protein structures: The structure and evolutionary dynamics of the globins journal January 1980
Direct, Real-Time Measurement of Rapid Inorganic Phosphate Release Using a Novel Fluorescent Probe and Its Application to Actomyosin Subfragment 1 ATPase journal July 1994
Molecular switch for signal transduction: structural differences between active and inactive forms of protooncogenic ras proteins journal February 1990
Deep mutational scanning: a new style of protein science journal July 2014
Collaboration gets the most out of software journal September 2013
Viewing Protein Fitness Landscapes Through a Next-Gen Lens journal October 2014
Allosteric Effects of the Oncogenic RasQ61L Mutant on Raf-RBD journal March 2015
An automated approach to network features of protein structure ensembles: An Automated Approach to Network Features of Protein Structure Ensembles journal September 2013
The Genomic and Cellular Foundations of Animal Origins journal November 2013
K-Ras protein as a drug target journal March 2016
Shift in the Equilibrium between On and Off States of the Allosteric Switch in Ras-GppNHp Affected by Small Molecules and Bulk Solvent Composition journal July 2012
A Comprehensive Survey of Ras Mutations in Cancer journal May 2012
The structural basis of the activation of Ras by Sos journal July 1998
Premetazoan genome evolution and the regulation of cell differentiation in the choanoflagellate Salpingoeca rosetta journal January 2013
The Structural Basis of Oncogenic Mutations G12, G13 and Q61 in Small GTPase K-Ras4B journal February 2016
How ras-related proteins talk to their effectors journal December 1996
Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding journal September 2015
The structural basis of the activation of Ras by Sos journal July 1998
A novel switch region regulates H-ras membrane orientation and signal output journal February 2008
The Ras-RasGAP Complex: Structural Basis for GTPase Activation and Its Loss in Oncogenic Ras Mutants journal July 1997
FastML: a web server for probabilistic reconstruction of ancestral sequences text January 2012
Structural Evidence for Feedback Activation by Ras·GTP of the Ras-Specific Nucleotide Exchange Factor SOS journal March 2003
Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding journal September 2015
Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli journal June 2009
Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1 journal July 2013

Cited By (21)

Deep generative models of genetic variation capture the effects of mutations journal September 2018
Data-driven supervised learning of a viral protease specificity landscape from deep sequencing and molecular simulations journal December 2018
Using deep mutational scanning to benchmark variant effect predictors and identify disease mutations journal July 2020
Extending chemical perturbations of the ubiquitin fitness landscape in a classroom setting reveals new constraints on sequence tolerance journal July 2018
Targeted insertional mutagenesis libraries for deep domain insertion profiling journal December 2019
Rhapsody: Pathogenicity prediction of human missense variants based on protein sequence, structure and dynamics posted_content August 2019
Targeted insertional mutagenesis libraries for deep domain insertion profiling journal December 2019
Using deep mutational scanning to benchmark variant effect predictors and identify disease mutations posted_content April 2020
MaveDB: an open-source platform to distribute and interpret data from multiplexed assays of variant effect journal November 2019
Understanding molecular mechanisms in cell signaling through natural and artificial sequence variation journal December 2018
Integration of Multi-level Molecular Scoring for the Interpretation of RAS-Family Genetic Variation posted_content October 2019
The reactivity-driven biochemical mechanism of covalent KRASG12C inhibitors journal May 2018
Modulating the cellular context broadly reshapes the mutational landscape of a model enzyme posted_content November 2019
Leveraging implicit knowledge in neural networks for functional dissection and engineering of proteins journal May 2019
Targeted insertional mutagenesis libraries for deep domain insertion profiling journal November 2019
Rhapsody: Pathogenicity prediction of human missense variants based on protein sequence, structure and dynamics posted_content August 2019
Extending chemical perturbations of the ubiquitin fitness landscape in a classroom setting reveals new constraints on sequence tolerance journal July 2018
Isoform-Specific Destabilization of the Active Site Reveals a Molecular Mechanism of Intrinsic Activation of KRas G13D journal August 2019
Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification cross-talk journal April 2018
Fitness of unregulated human Ras mutants modeled by implementing computational mutagenesis and machine learning techniques journal June 2019
Multiplexed assays of variant effects contribute to a growing genotype–phenotype atlas journal August 2018

Similar Records

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Life Sciences & Biomedicine - Other Topics