Cited by 133Protein Kinase Regulation and GTPase SignalingCellular transport and secretionReceptor Mechanisms and Signaling

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The small GTPases K-Ras, N-Ras, and H-Ras have distinct biochemical properties determined by allosteric effects

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Johnson, Christian W., Reid, Derion, Parker, Jillian A., Salter, Shores, Knihtila, Ryan, Kuzmic, Petr, Mattos, Carla·2017·Journal of Biological Chemistry

DOI 10.1074/jbc.m117.778886PMC PMC5546037

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Abstract

H-Ras, K-Ras, and N-Ras are small GTPases that are important in the control of cell proliferation, differentiation, and survival, and their mutants occur frequently in human cancers. The G-domain, which catalyzes GTP hydrolysis and mediates downstream signaling, is 95% conserved between the Ras isoforms. Because of their very high sequence identity, biochemical studies done on H-Ras have been considered representative of all three Ras proteins. We show here that this is not a valid assumption. Using enzyme kinetic assays under identical conditions, we observed clear differences between the three isoforms in intrinsic catalysis of GTP by Ras in the absence and presence of the Ras-binding domain (RBD) of the c-Raf kinase protein (Raf-RBD). Given their identical active sites, isoform G-domain differences must be allosteric in origin, due to remote isoform-specific residues that affect conformational states. We present the crystal structure of N-Ras bound to a GTP analogue and interpret the kinetic data in terms of structural features specific for H-, K-, and N-Ras.

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BackgroundInfluential
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MethodInfluential
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Result
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BackgroundMethodInfluentialSelf-citation
Ras trafficking, localization and compartmentalized signalling
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Background
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BackgroundResultInfluential
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Background
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Background
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Self-citation
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Background
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Background
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BackgroundSelf-citation
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Organic extraction of Pi with isobutanol/toluene
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BackgroundMethodInfluential
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BackgroundSelf-citation
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Background
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Background
Two conformational states of Ras GTPase exhibit differential GTP-binding kinetics
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Self-citation
Characterization of a 78-residue fragment of c-Raf-1 that comprises a minimal binding domain for the interaction with Ras-GTP.
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Background
[18] Intrinsic and GTPase-activating protein-stimulated Ras GTPase assays
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BackgroundMethodInfluential
Distinct dynamics and interaction patterns in H- and K-Ras oncogenic P-loop mutants: Interaction Patterns in Ras Oncogenic Mutants
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BackgroundResult
Structure of a Transient Intermediate for GTP Hydrolysis by Ras
Ford, B. A., Horn̆ák, V., Kleinman, H., & Nassar, N. N. (2006) · Structure · doi:10.1016/j.str.2005.12.010 · Cited by 52
Switching Nucleotide Specificity of Ha-Ras p21 by a Single Amino Acid Substitution at Aspartate 119
Zhong, J.-M., Chen‐Hwang, M., & Hwang, Y. (1995) · Journal of Biological Chemistry · doi:10.1074/jbc.270.17.10002 · Cited by 52 · cited 1×
“The allosteric lobe contains the NKXD (residues 116 – 119) motif responsible for the specificity of the guanine nucleotide (11), and the EXSAK (residues 143–147) motif, aided by effector lobe residue Phe-28, helps to stabilize nucleotide binding.”
Background
Real-time NMR Study of Three Small GTPases Reveals That Fluorescent 2′(3′)-O-(N-Methylanthraniloyl)-tagged Nucleotides Alter Hydrolysis and Exchange Kinetics
Mazhab‐Jafari, M. T., Marshall, C. B., Smith, M. J., Gasmi-Seabrook, G. M. C., Stambolic, V., Rottapel, R. K., Neel, B. G., & Ikura, M. (2009) · Journal of Biological Chemistry · doi:10.1074/jbc.c109.064766 · Cited by 49 · cited 2×
“However, the mant nucleotides have recently been shown by NMR to alter the active site environment in unpredictable ways (57).”
BackgroundResult
Tyrosine phosphorylation of RAS by ABL allosterically enhances effector binding
Ting, P. Y., Johnson, C. W., Fang, C., Cao, X., Graeber, T. G., Mattos, C., & Colicelli, J. (2015) · The FASEB Journal · doi:10.1096/fj.15-271510 · Cited by 45 · cited 1×
“Single-turnover experiments were performed to obtain rate constants for hydrolysis of GTP catalyzed by Ras, using a discontinuous assay based on the production of (32)Pi, as described previously (27, 35).”
MethodSelf-citation
Intrinsic K-Ras dynamics: A novel molecular dynamics data analysis method shows causality between residue pair motions
Vatansever, S., Gumus, Z. H., & Erman, B. (2016) · Scientific Reports · doi:10.1038/srep37012 · Cited by 45 · cited 2×
“Although the Raf-RBD binds only at switch I, the two switches are conformationally linked, with helix 3 and switch II driving the conformation of switch I (71).”
Background
DRoP: A Water Analysis Program Identifies Ras-GTP-Specific Pathway of Communication between Membrane-Interacting Regions and the Active Site
Kearney, B. M., Johnson, C. W., Roberts, D. M., Swartz, P., & Mattos, C. (2013) · Journal of Molecular Biology · doi:10.1016/j.jmb.2013.10.036 · Cited by 43 · cited 7×
“Protein expression and purification of wild-type Ras isoforms was performed as described for H-Ras (27, 77), with no modifications for K-Ras or N-Ras.”
BackgroundMethodInfluentialSelf-citation
Neutron Crystal Structure of RAS GTPase Puts in Question the Protonation State of the GTP γ-Phosphate
Knihtila, R., Holzapfel, G. A., Weiss, K. L., Meilleur, F., & Mattos, C. (2015) · Journal of Biological Chemistry · doi:10.1074/jbc.m115.679860 · Cited by 39 · cited 2×
“and the NKXD and EXSAK motifs that directly affect nucleotide binding (loop 8, helix 4 and helix 5) (10).”
BackgroundSelf-citation
Characterization of recombinant human Kirsten-ras (4B) p21 produced at high levels in Escherichia coli and insect baculovirus expression systems
Lowe, P. N., Pagé, M., Bradley, S., Rhodes, S., Sydenham, M. A., Paterson, H., & Skinner, R. H. (1991) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)52347-4 · Cited by 37
Stereochemistry of the elongation factor Tu . GTP complex
LEUPOLD, C. M., Goody, R. S., & Wittinghofer, A. (1983) · European Journal of Biochemistry · doi:10.1111/j.1432-1033.1983.tb07643.x · Cited by 36 · cited 1×
“The original hydrolysis experiments published for H-Ras in the late 1980s used a [ -(32)P]GTP discontinuous assay, referencing a 1983 paper on determination of GTP hydrolysis rate constants for EF-Tu, in which hydrolysis was carried out for 60 min with time points collected every minute (67).”
Method
Shift in the Equilibrium between On and Off States of the Allosteric Switch in Ras-GppNHp Affected by Small Molecules and Bulk Solvent Composition
Holzapfel, G. A., Buhrman, G., & Mattos, C. (2012) · Biochemistry · doi:10.1021/bi300509j · Cited by 35 · cited 1×
“previously described (26, 77), and concentrated to 3.”
BackgroundSelf-citation
Interaction of GTPase-activating protein with p21ras, measured using a continuous assay for inorganic phosphate release
Webb, M. R., & Hunter, J. L. (1992) · Biochemical Journal · doi:10.1042/bj2870555 · Cited by 33
[2] Parameter estimates from nonlinear models
Watts, D. G. (1994) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/s0076-6879(94)40041-5 · Cited by 30
Decoding RAS isoform and codon-specific signalling
Newlaczyl, A. U., Hood, F. E., Coulson, J. M., & Prior, I. A. (2014) · Biochemical Society Transactions · doi:10.1042/bst20140057 · Cited by 20 · cited 1×
“They could impact isoform-specific biological functions in ways that have not yet been determined (73).”
Background
Expression, purification, crystallization and X-ray data collection for RAS and its mutants
Johnson, C. W., Buhrman, G., Ting, P. Y., Colicelli, J., & Mattos, C. (2015) · Data in Brief · doi:10.1016/j.dib.2015.12.007 · Cited by 19 · cited 2×
“previously described (26, 77), and concentrated to 3.”
BackgroundMethodSelf-citation
Role of Magnesium in Nucleotide Exchange on the Small G Protein Rac Investigated Using Novel Fluorescent Guanine Nucleotide Analogues
Shutes, A., Phillips, R. A., Corrie, J. E. T., & Webb, M. R. (2002) · Biochemistry · doi:10.1021/bi0119464 · Cited by 17 · cited 1×
“Enzyme-coupled reactions provide an alternative (59, 60, 63, 64), but this method reports on GTPase product release rather than on cleavage of the phosphodiester bond (64, 65).”
Background
State 1(T) Inhibitors of Activated Ras
Kalbitzer, H. R., & Spoerner, M. (2013) · The Enzymes · doi:10.1016/b978-0-12-416749-0.00004-x · Cited by 17
Inhibitors of the Ras Superfamily G-proteins, Part A
(2013) · The Enzymes · doi:10.1016/c2012-0-03660-5 · Cited by 1

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See all 133

Authors: Johnson, C. W., Reid, D., Parker, J. A., Salter, S., Knihtila, R., Kuzmic, P., & Mattos, C.
Affiliations: Northeastern University (US)
Corresponding author: Mattos, Carla
ORCID: Johnson, Christian W.
Parker, Jillian A.
Mattos, Carla
Venue: Journal of Biological Chemistry
Publisher: Elsevier BV
Published: 2017-08
Volume: 292
Issue: 31
Pages: 12981-12993
ISSN: 0021-9258 (ISSN-L)
DOI: 10.1074/jbc.m117.778886
PMC: PMC5546037
OpenAlex: W2717068414
Cited by: 133
References: 84
Citations by year: 2026: 12 · 2025: 7 · 2024: 15 · 2023: 10 · 2022: 20 · 2021: 18 · 2020: 14 · 2019: 20 · 2018: 12 · 2017: 5
FWCI: 5.69
Type: journal article
Language: en
Open access: hybrid
doi.org
Licence: cc-by
www.elsevier.com/tdm/userlicense/1.0/ (tdm)
creativecommons.org/licenses/by/4.0/ (vor, 1129 d)
Locations: Journal of Biological Chemistry (publishedVersion, cc-by)
Europe PMC (PubMed Central) (publishedVersion)
PubMed Central (publishedVersion)
PubMed (publishedVersion)
Funders: National Science Foundation (1517295)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
Keywords: Ras GTPase, KRAS, NRAS, H-Ras, Ras isoforms, allosteric regulation, GTP hydrolysis, enzyme kinetics, Ras-binding domain
MeSH terms: Allosteric Regulation, Allosteric Site, Enzyme Stability, Guanosine Triphosphate, Humans, Isoenzymes, Ligands, Membrane Proteins, Models, Molecular, Peptide Fragments, Protein Conformation, Recombinant Proteins, Dinucleoside Phosphates, Proto-Oncogene Proteins p21(ras), Point Mutation, Crystallography, X-Ray, Proto-Oncogene Proteins c-raf, Amino Acid Substitution, Catalytic Domain, GTP Phosphohydrolases, Protein Interaction Domains and Motifs, Biocatalysis

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The small GTPases K-Ras, N-Ras, and H-Ras have distinct biochemical properties determined by allosteric effects

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