The small GTPases K-Ras, N-Ras, and H-Ras have distinct biochemical properties determined by allosteric effects
Open accessAbstract
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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RAS mutants were once thought to be locked in the GTP-bound on state and undruggable, but detailed structural and biochemical analyses have revealed differences among RAS mutants in GTPase activity, GDP-GTP exchange and effector binding.
“RAS mutants were once viewed as being ‘locked’ in the GTP-bound ‘on’ state, impervious to GAP-stimulated or intrinsic GTP hydrolysis, and hence undruggable. However, detailed structural and biochemical analyses of various RAS mutants associated with cancer and ‘RASopathies’”
From The current state of the art and future trends in RAS-targeted cancer therapies (2022) · cites this paper as [45]
At the cellular level, isoform specificity to effector proteins is primarily determined via membrane interactions, but differences among RAS isoforms' absolute effector protein binding affinities rise from allosteric effects.
“At the cellular level, the isoform specificity to effector proteins is primarily determined via membrane interactions [24], but the differences among RAS isoforms’ absolute effector protein binding affinities rise from allosteric effects [25].”
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The Complexity of the ERK/MAP-Kinase Pathway and the Treatment of Melanoma Skin Cancer
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Organic extraction of Pi with isobutanol/toluene
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Jang, H., Banerjee, A., Chavan, T., Lu, S., Zhang, J., Gaponenko, V., & Nussinov, R. (2015) · The FASEB Journal · doi:10.1096/fj.15-279091 · Cited by 75 · cited 1× · see citing works
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Background[18] Intrinsic and GTPase-activating protein-stimulated Ras GTPase assays
Bollag, G. E., & McCormick, F. P. (1995) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/s0076-6879(95)55020-8 · Cited by 55 · cited 4× · see citing works
“The (32)Pi product was isolated using an organic extraction method (36, 37), and the radioactivity was measured with a scintillation counter (300SL, LabLogic).”
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Differential dynamics of RAS isoforms in GDP- and GTP-bound states
Kapoor, A., & Travesset, A. (2015) · Proteins Structure Function and Bioinformatics · doi:10.1002/prot.24805 · Cited by 53 · cited 2× · see citing works
“However, they are modulated differently by surrounding residues in H-, K-, and N-Ras, leading to differences in dynamics (6, 34) and kinetics of GTP hydrolysis as observed in this study.”
BackgroundResultStructure 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 · see citing works
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× · see citing works
“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.”
BackgroundMazhab‐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× · see citing works
“However, the mant nucleotides have recently been shown by NMR to alter the active site environment in unpredictable ways (57).”
BackgroundResultTyrosine 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 46 · cited 1× · see citing works
“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-citationVatansever, S., Gumus, Z. H., & Erman, B. (2016) · Scientific Reports · doi:10.1038/srep37012 · Cited by 45 · cited 2× · see citing works
“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).”
BackgroundKearney, 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× · see citing works
“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-citationKnihtila, R., Holzapfel, G. A., Weiss, K. L., Meilleur, F., & Mattos, C. (2015) · Journal of Biological Chemistry · doi:10.1074/jbc.m115.679860 · Cited by 40 · cited 2× · see citing works
“and the NKXD and EXSAK motifs that directly affect nucleotide binding (loop 8, helix 4 and helix 5) (10).”
BackgroundSelf-citationLowe, 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 · see citing works
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× · see citing works
“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).”
MethodHolzapfel, G. A., Buhrman, G., & Mattos, C. (2012) · Biochemistry · doi:10.1021/bi300509j · Cited by 35 · cited 1× · see citing works
“previously described (26, 77), and concentrated to 3.”
BackgroundSelf-citationWebb, M. R., & Hunter, J. L. (1992) · Biochemical Journal · doi:10.1042/bj2870555 · Cited by 33 · see citing works
[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 · see citing works
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 19 · cited 1× · see citing works
“They could impact isoform-specific biological functions in ways that have not yet been determined (73).”
BackgroundExpression, 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× · see citing works
“previously described (26, 77), and concentrated to 3.”
BackgroundMethodSelf-citationShutes, A., Phillips, R. A., Corrie, J. E. T., & Webb, M. R. (2002) · Biochemistry · doi:10.1021/bi0119464 · Cited by 17 · cited 1× · see citing works
“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).”
BackgroundState 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 · see citing works
Inhibitors of the Ras Superfamily G-proteins, Part A
(2013) · The Enzymes · doi:10.1016/c2012-0-03660-5 · Cited by 1 · see citing works
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The Frequency of Ras Mutations in Cancer
Prior, I. A., Hood, F. E., & Hartley, J. L. (2020) · Cancer Research · doi:10.1158/0008-5472.can-19-3682 · Cited by 1,130 · see citing works
“Isoformspecific signaling is thought to be mediated by differential intracellular localisation that favors preferential coupling to specific effector pathways (4, 22, 23), and by distinct biochemical properties imparted by allosteric lobe sequence variations between each isoform (24).”
BackgroundMolecular Dynamics Simulations in Drug Discovery and Pharmaceutical Development
Salo‐Ahen, O. M. H., Alanko, I., Bhadane, R. N., Bonvin, A. M. J. J., Honorato, R. V., Hossain, S., Juffer, A. H., Kabedev, A., Lahtela-Kakkonen, M. K., Larsen, A. S., et al. (2020) · Processes · doi:10.3390/pr9010071 · Cited by 693 · see citing works
The current state of the art and future trends in RAS-targeted cancer therapies
Punekar, S. R., Velcheti, V., Neel, B. G., & Wong, K.-K. (2022) · Nature Reviews Clinical Oncology · doi:10.1038/s41571-022-00671-9 · Cited by 549 · see citing works
The current understanding of KRAS protein structure and dynamics
Pantsar, T. (2019) · Computational and Structural Biotechnology Journal · doi:10.1016/j.csbj.2019.12.004 · Cited by 288 · see citing works
“Since, it has been confirmed that the different isoforms exhibit distinct biochemical profiles [91].”
BackgroundSHP2 inhibition diminishes KRASG12C cycling and promotes tumor microenvironment remodeling
Fedele, C., Li, S., Teng, K. W., Foster, C. J. R., Peng, D., Ran, H., Mita, P., Geer, M. J., Hattori, T., Koide, A., et al. (2021) · Journal of Experimental Medicine · doi:10.1084/jem.20201414 · Cited by 265 · see citing works
“” More recent analyses revealed subtle but key differences between RAS mutants in intrinsic and residual GAP-catalyzed GTPase activity, intrinsic and SOS-stimulated exchange, and effector binding (Bandaru et al., 2017; Gremer et al., 2011; Hunter et al., 2015; Johnson et al., 2017; Mazhab-Jafari et al., 2015; Smith an…”
BackgroundSerebriiskii, I. G., Connelly, C., Frampton, G., Newberg, J., Cooke, M., Miller, V., Ali, S., Ross, J. S., Handorf, E., Arora, S., et al. (2019) · Nature Communications · doi:10.1038/s41467-019-11530-0 · Cited by 215 · see citing works
Chen, J., Zhang, S., Wang, W., Pang, L., Zhang, Q., & Liu, X. (2021) · Journal of Chemical Information and Modeling · doi:10.1021/acs.jcim.0c01470 · Cited by 140 · see citing works
Deconstruction of the Ras switching cycle through saturation mutagenesis
Bandaru, P., Shah, N. H., Bhattacharyya, M., Barton, J. P., Kondo, Y., Cofsky, J. C., Gee, C. L., Chakraborty, A. K., Kortemme, T., Ranganathan, R., et al. (2017) · eLife · doi:10.7554/elife.27810 · Cited by 117 · see citing works
“The sequences of the three principal isoforms of Ras (H-Ras, K-Ras and N-Ras) are almost invariant in vertebrates, and Ras proteins are highly conserved across all metazoans (Figure 1—figure supplement 1 shows a phylogenetic tree based on alignment of H-Ras orthologs) (Johnson et al., 2017).”
BackgroundNussinov, R., Jang, H., Tsai, C., & Cheng, F. (2019) · PLoS Computational Biology · doi:10.1371/journal.pcbi.1006658 · Cited by 129 · see citing works
Pantsar, T., Rissanen, S., Dauch, D., Laitinen, T., Vattulainen, I., & Poso, A. (2018) · PLOS Computational Biology · doi:10.1371/journal.pcbi.1006458 · Cited by 85 · see citing works
“At the cellular level, the isoform specificity to effector proteins is primarily determined via membrane interactions [24], but the differences among RAS isoforms’ absolute effector protein binding affinities rise from allosteric effects [25].”
BackgroundOncogenic G12D mutation alters local conformations and dynamics of K-Ras
Vatansever, S., Erman, B., & Gumus, Z. H. (2019) · Scientific Reports · doi:10.1038/s41598-019-48029-z · Cited by 93 · see citing works
Johnson, C. W., Lin, Y.-J., Reid, D., Parker, J., Pavlopoulos, S., Dischinger, P., Graveel, C., Aguirre, A. J., Steensma, M., Haigis, K. M., et al. (2019) · Cell Reports · doi:10.1016/j.celrep.2019.07.026 · Cited by 89 · see citing works
“We were unable to crystalize NRas G13D bound to GDP. Hydrolysis assays to determine rate constants—Hydrolysis of GTP was measured by production of radioactive inorganic phosphate from γ32P-GTP (Perkin Elmer) loaded Ras proteins, as previously published (Johnson et al., 2017).”
BackgroundMethodInfluentialSelf-citationMo, S. P., Coulson, J. M., & Prior, I. A. (2018) · Biochemical Society Transactions · doi:10.1042/bst20180173 · Cited by 85 · see citing works
The Ras switch in structural and historical perspective
Gasper, R., & Wittinghofer, F. (2019) · Biological Chemistry · doi:10.1515/hsz-2019-0330 · Cited by 88 · see citing works
“Some biochemical properties are slightly different between the isoforms, i.e. the intrinsic GTPase, which may potentially be significant (Johnson et al., 2017).”
BackgroundKRAS G12C Mutations in NSCLC: From Target to Resistance
Addeo, A., Banna, G. L., & Friedlaender, A. (2021) · Cancers · doi:10.3390/cancers13112541 · Cited by 84 · see citing works
“Some may be associated with specific downstream effector pathways, influencing the impact of each isoform[25,26].”
BackgroundParker, J. A., Volmar, A. Y., Pavlopoulos, S., & Mattos, C. (2018) · Structure · doi:10.1016/j.str.2018.03.018 · Cited by 81 · see citing works
Self-citationLanfredini, S., Thapa, A., & O’Neill, E. E. (2019) · Biochemical Society Transactions · doi:10.1042/bst20170521 · Cited by 78 · see citing works
The Interdependent Activation of Son-of-Sevenless and RasPreprint
Bandaru, P., Kondo, Y., & Kuriyan, J. (2018) · Cold Spring Harbor Perspectives in Medicine · doi:10.1101/cshperspect.a031534 · Cited by 78 · see citing works
“The three principal isoforms of Ras (H-Ras, K-Ras, and N-Ras) are generally highly conserved in metazoans (Rojas et al. 2012; Johnson et al. 2017), though less so in invertebrates compared to vertebrates (Bandaru et al. 2017).”
BackgroundCookis, T., & Mattos, C. (2021) · Biomolecules · doi:10.3390/biom11070996 · Cited by 68 · see citing works
“This motif is followed by loop 8, containing residue R123 which makes a salt bridge interaction with E143 of the ExSAK motif (residues 143–147), linking two highly conserved regions important for active site stability [52].”
BackgroundSelf-citationThe structural basis for Ras activation of PI3Kα lipid kinase
Zhang, M., Jang, H., & Nussinov, R. (2019) · Physical Chemistry Chemical Physics · doi:10.1039/c9cp00101h · Cited by 67 · see citing works
Wang, Y., Ji, D., Lei, C., Chen, Y., Qiu, Y., Li, X., Li, M., Ni, D., Pu, J., Zhang, J., et al. (2021) · Computational and Structural Biotechnology Journal · doi:10.1016/j.csbj.2021.01.044 · Cited by 64 · see citing works
“There are four isoforms of Ras in human cells: H-Ras, N-Ras, K-Ras4A and K-Ras4B (splice variants of gene KRAS ) [11].”
BackgroundParker, J. A., & Mattos, C. (2017) · Cold Spring Harbor Perspectives in Medicine · doi:10.1101/cshperspect.a031427 · Cited by 60 · see citing works
“3D) (Johnson et al. 2017).”
BackgroundInfluentialSelf-citationProtCID: a data resource for structural information on protein interactions
Xu, Q., & Dunbrack, R. L. (2020) · Nature Communications · doi:10.1038/s41467-020-14301-4 · Cited by 60 · see citing works
“With ProtCID, we have identi fi ed an α 4 – α 5 dimer in a crystal of GNP-bound NRAS (PDB: 5UHV [https://doi.org/10.2210/pdb5UHV/ pdb]), that was unrecognized by the authors of this structure 50 .”
BackgroundInfluentialRaf promotes dimerization of the Ras G-domain with increased allosteric connections
Packer, M. R., Parker, J. A., Chung, J. K., Li, Z., Lee, Y. K., Cookis, T., Guterres, H., Alvarez, S. A., Hossain, M. A., Donnelly, D. P., et al. (2021) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2015648118 · Cited by 55 · see citing works
Self-citationKRAS Switch Mutants D33E and A59G Crystallize in the State 1 Conformation
Lu, J., Bera, A. K., Gondi, S. R., & Westover, K. D. (2017) · Biochemistry · doi:10.1021/acs.biochem.7b00974 · Cited by 50 · see citing works
Enhanced BRAF engagement by NRAS mutants capable of promoting melanoma initiation
Murphy, B., Terrell, E. M., Chirasani, V. R., Weiss, T. J., Lew, R. E., Holderbaum, A. M., Dhakal, A., Posada, V., Fort, M., Bodnar, M. S., et al. (2022) · Nature Communications · doi:10.1038/s41467-022-30881-9 · Cited by 49 · see citing works
Structure of the MRAS–SHOC2–PP1C phosphatase complex
Hauseman, Z. J., Fodor, M., Dhembi, A., Viscomi, J. S., Egli, D., Bleu, M., Katz, S., Park, E., Jang, D. M., Porter, K. A., et al. (2022) · Nature · doi:10.1038/s41586-022-05086-1 · Cited by 47 · see citing works
Implications of farnesyltransferase and its inhibitors as a promising strategy for cancer therapy
Klochkov, S. G., Евгеньевна, Н. М., Yarla, N. S., Parvathaneni, M., Sharma, B., Tarasov, V. V., Barreto, G. E., Бачурин, С. О., Ashraf, G. M., & Aliev, G. M. (2017) · Seminars in Cancer Biology · doi:10.1016/j.semcancer.2017.10.010 · Cited by 46 · see citing works
Kumar, S., Singh, J., Narasimhan, B., Shah, S. A. A., Lim, S. M., Ramasamy, K., & Mani, V. (2018) · Chemistry Central Journal · doi:10.1186/s13065-018-0475-5 · Cited by 42 · see citing works
Burge, R. A., & Hobbs, G. A. (2022) · Advances in cancer research · doi:10.1016/bs.acr.2021.07.004 · Cited by 44 · see citing works
Killoran, R. C., & Smith, M. J. (2019) · Journal of Biological Chemistry · doi:10.1074/jbc.ra119.008653 · Cited by 41 · see citing works
“There has been little attempt to study differences in nucleotide exchange and/or GTP hydrolysis rates for the RAS isoforms in their individual contexts; however, one study found that the intrinsic rates of GTP catalysis differed across isoforms using [(32)Pi]GTP single turnover (29).”
Ras assemblies and signaling at the membrane
Nussinov, R., Tsai, C., & Jang, H. (2020) · Current Opinion in Structural Biology · doi:10.1016/j.sbi.2020.01.009 · Cited by 41 · see citing works
Structural Learning of Proteins Using Graph Convolutional Neural Networks
Zamora-Resendiz, R., & Crivelli, S. (2019) · doi:10.1101/610444 · Cited by 37 · see citing works
“This value is inline with the number of unique structural partitions, which were referenced from Mattos et al.’s studies [50, 51].”
BackgroundInfluentialCastelli, M., Marchetti, F., Osuna, S., Oliveira, A. S. F., Mulholland, A. J., Serapian, S. A., & Colombo, G. (2023) · Journal of the American Chemical Society · doi:10.1021/jacs.3c11396 · Cited by 38 · see citing works
“37,38,40,45,54 Still, while switches I and II remain expectedly very mobile in the GTP-active state, we find that they both retain mild allosteric coupling to the G domain in catalytically crucial regions (cf. Figure 2 bottom): Mg 2+ -bound Thr35 in the former case, and Thr58/Ala59/[Gln61] in the…”
InfluentialAllostery and dynamics in small G proteins
Mott, H. R., & Owen, D. (2018) · Biochemical Society Transactions · doi:10.1042/bst20170569 · Cited by 37 · see citing works
Zhang, H., Ni, D., Fan, J., Li, M.-Y., Zhang, J., Chen, H., Nussinov, R., & Lu, S. (2022) · Journal of Chemical Information and Modeling · doi:10.1021/acs.jcim.2c00591 · Cited by 36 · see citing works
Rahimova, R., Fontanel, S., Lionne, C., Jordheim, L. P., Peyrottes, S., & Chaloin, L. (2018) · PLoS Computational Biology · doi:10.1371/journal.pcbi.1005943 · Cited by 35 · see citing works
Prediction of allosteric communication pathways in proteins
Haliloǧlu, T., Hacisuleyman, A., & Erman, B. (2022) · Bioinformatics · doi:10.1093/bioinformatics/btac380 · Cited by 34 · see citing works
Structure of an inactive conformation of GTP-bound RhoA GTPase
Lin, Y., Lu, S., Zhang, J., & Zheng, Y. (2021) · Structure · doi:10.1016/j.str.2020.12.015 · Cited by 27 · see citing works
Dynamically encoded reactivity of Ras enzymes: opening new frontiers for drug discovery
Pálfy, G., Menyhárd, D. K., & Perczel, A. (2020) · Cancer and Metastasis Reviews · doi:10.1007/s10555-020-09917-3 · Cited by 26 · see citing works
Zhao, J., Zhang, T., Liang, Y., Zou, H., & Zhang, J. (2021) · Food and Chemical Toxicology · doi:10.1016/j.fct.2021.112411 · Cited by 26 · see citing works
Chao, F.-A., Dharmaiah, S., Taylor, T. E., Messing, S. A. J., Gillette, W., Esposito, D., Nissley, D. V., McCormick, F. P., Byrd, R. A., Simanshu, D. K., et al. (2022) · Journal of the American Chemical Society · doi:10.1021/jacs.2c00007 · Cited by 26 · see citing works
Bao, H., Wang, W., Sun, H., & Chen, J. (2023) · Journal of Enzyme Inhibition and Medicinal Chemistry · doi:10.1080/14756366.2023.2195995 · Cited by 26 · see citing works
Zeng, J., Weng, J., Zhang, Y., Xia, F., Cui, Q., & Xu, X. (2021) · The Journal of Physical Chemistry B · doi:10.1021/acs.jpcb.1c04679 · Cited by 25 · see citing works
RAS‐targeted cancer therapy: Advances in drugging specific mutations
Liu, C., Ye, D., Yang, H., Chen, X., Su, Z., Li, X., Ding, M., & Liu, Y. (2023) · MedComm · doi:10.1002/mco2.285 · Cited by 24 · see citing works
Dynamic conformational equilibria in the active states of KRAS and NRAS
Rennella, E., Henry, C., Dickson, C. J., Georgescauld, F., Wales, T. E., Erdmann, D., Cotesta, S., Maira, S.-M., Sedrani, R. C., Brachmann, S. M., et al. (2024) · RSC Chemical Biology · doi:10.1039/d4cb00233d · Cited by 24 · see citing works
Deep phylogeny of cancer drivers and compensatory mutations
Rochman, N. D., Wolf, Y. I., & Koonin, E. V. (2020) · Communications Biology · doi:10.1038/s42003-020-01276-7 · Cited by 24 · see citing works
Girard, É., Lopes, P., Spoerner, M., Dhaussy, A., Prangé, T., Kalbitzer, H. R., & Colloc’h, N. (2022) · Chemical Science · doi:10.1039/d1sc05488k · Cited by 23 · see citing works
Meinohl, C., Barnard, S. J., Fritz‐Wolf, K., Unger, M., Porr, A., Heipel, M., Wirth, S., Madlung, J., Nordheim, A., Menke, A., et al. (2019) · Cancers · doi:10.3390/cancers12010030 · Cited by 23 · see citing works
“The bound nucleotide in Ras changes the confirmation of the protein and its properties to interact with effector and regulatory proteins [23,38].”
BackgroundLópez, C. A., Zhang, X., Aydin, F., Shrestha, R., Van, Q. N., Stanley, C. B., Carpenter, T. S., Nguyen, K., Patel, L. A., Chen, D., et al. (2022) · Journal of Chemical Theory and Computation · doi:10.1021/acs.jctc.2c00168 · Cited by 18 · see citing works
Direct GDP-KRAS G12C inhibitors and mechanisms of resistance: the tip of the iceberg
Rosen, J. C., Sacher, A. G., & Tsao, M. (2023) · Therapeutic Advances in Medical Oncology · doi:10.1177/17588359231160141 · Cited by 22 · see citing works
Sharma, A. K., Pei, J., Yang, Y., Dyba, M. D., Smith, B. D., Rabara, D., Larsen, E. K., Lightstone, F. C., Esposito, D., Stephen, A. G., et al. (2024) · Journal of Biological Chemistry · doi:10.1016/j.jbc.2024.105650 · Cited by 24 · see citing works
“Growing evidence, however, suggests that KRAS and HRAS behave differently and exhibit mutant-speci fi c distinct conformational features (7, 15, 16).”
BackgroundFuentes‐Mateos, R., Jimeno, D., Gómez, C., Calzada, N., Fernández‐Medarde, A., & Santos, E. (2019) · Cell Death and Disease · doi:10.1038/s41419-019-2075-2 · Cited by 20 · see citing works
Donohue, E., Khorsand, S., Mercado, G., Varney, K. M., Wilder, P. T., Yu, W., MacKerell, A. D., Alexander, P., Van, Q. N., Moree, B., et al. (2019) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1905516116 · Cited by 20 · see citing works
“To identify KRas ligands using SHG, we targeted recombinant His-tagged KRas4b (1-166) carrying the G12D mutation (hereafter referred to as KRas).”
BackgroundMethodZhu, Z., Ma, G., Yang, M., Tan, C., Yang, G., Wang, S., Li, N., Ge, F., & Wang, S. (2021) · Environmental Microbiology · doi:10.1111/1462-2920.15626 · Cited by 19 · see citing works
Extracting phylogenetic dimensions of coevolution reveals hidden functional signals
Colavin, A., Atolia, E., Bitbol, A., & Huang, K. C. (2022) · Scientific Reports · doi:10.1038/s41598-021-04260-1 · Cited by 19 · see citing works
“The Ras superfamily of membrane-associated small G-proteins is highly conserved and controls a broad range of cellular processes 66 , has inactive and active states that are regulated by a GTPase-activated protein 67 , and has been implicated in cancer 68 .”
BackgroundBao, H., Wang, W., Sun, H. B., & Chen, J. Z. (2023) · SAR and QSAR in environmental research · doi:10.1080/1062936x.2023.2165542 · Cited by 19 · see citing works
Hurtado‐Navarro, L., Cuenca‐Zamora, E. J., Zamora, L., Bellosillo, B., Such, E., Soler‐Espejo, E., Martínez‐Banaclocha, H., Hernández‐Rivas, J. M., Marco, J., Martínez-Alarcón, L., et al. (2023) · Cell Reports Medicine · doi:10.1016/j.xcrm.2023.101329 · Cited by 17 · see citing works
“Mutations in key residues in exon 2 or exon 3 (positions 10–14 [P loop region] and 58–72 [Switch-II region]) affect the GTP-binding site, preventing the conversion of the active RAS-GTP into inactive RAS-GDP, 35,36 leading to the continued activation of its downstream targets MEK1/2 and ERK1/2, 37 promoting cell proli…”
BackgroundBehairy, M. Y., Soltan, M., Adam, M., Refaat, A., Ezz, E. M., Albogami, S. M., Fayad, E., Althobaiti, F., Gouda, A., Sileem, A. E., et al. (2022) · Frontiers in Genetics · doi:10.3389/fgene.2022.872845 · Cited by 14 · see citing works
“The active site for GTP-binding is formed via the P-loop, Switch-I, and switch-II with residue ranges of 10 – 17, 30 – 40, and 60 – 76 amino acids, respectively (Johnson et al., 2017).”
BackgroundWater Distribution within Wild-Type NRas Protein and Q61 Mutants during Unrestrained QM/MM Dynamics
Tichauer, R. H., Favre, G., Cabantous, S., Landa, G., Hémeryck, A., & Brut, M. (2018) · Biophysical Journal · doi:10.1016/j.bpj.2018.07.042 · Cited by 16 · see citing works
“12–13, 32, 35, and 60–61 from Ras, Arg 789 from GAP, GTP, and a Mg2þ ion with two coordinated water molecules, found in the crystallographic structures of Ras (12,21,39,46) (see Fig.”
BackgroundZeng, J., Chen, J., Xia, F., Cui, Q., Deng, X., & Xu, X. (2022) · Physical Chemistry Chemical Physics · doi:10.1039/d2cp00274d · Cited by 16 · see citing works
Colorectal Cancer Modeling with Organoids: Discriminating between Oncogenic RAS and BRAF Variants
Post, J. B., Roodhart, J. M. L., & Snippert, H. J. G. (2020) · Trends in cancer · doi:10.1016/j.trecan.2019.12.005 · Cited by 16 · see citing works
Allosteric site variants affect GTP hydrolysis on Ras
Johnson, C. W., Fetics, S., Davis, K. P., Rodrigues, J. A., & Mattos, C. (2023) · Protein Science · doi:10.1002/pro.4767 · Cited by 15 · see citing works
Self-citationHu, F., Wang, Y., Zeng, J., Deng, X., Xia, F., & Xu, X. (2024) · The Journal of Physical Chemistry B · doi:10.1021/acs.jpcb.3c07666 · Cited by 15 · see citing works
The Importance of Mg 2+ ‐Free State in Nucleotide Exchange of Oncogenic K‐Ras Mutants
Pálfy, G., Menyhárd, D. K., Ákontz‐Kiss, H., Vida, I., Batta, G., Tőke, O., & Perczel, A. (2022) · Chemistry - A European Journal · doi:10.1002/chem.202201449 · Cited by 14 · see citing works
Mechanisms of isoform-specific residue influence on GTP-bound HRas, KRas, and NRas
Volmar, A. Y., Guterres, H., Zhou, H., Reid, D. F., Pavlopoulos, S., Makowski, L., & Mattos, C. (2022) · Biophysical Journal · doi:10.1016/j.bpj.2022.07.005 · Cited by 14 · see citing works
Self-citationKinetic and thermodynamic allostery in the Ras protein family
Manley, L. J., & Lin, M. M. (2023) · Biophysical Journal · doi:10.1016/j.bpj.2023.08.010 · Cited by 14 · see citing works
Reder, H., Wagner, S., Gamerdinger, U., Sandmann, S., Wuerdemann, N., Braeuninger, A., Dugas, M., Gattenloehner, S., Klußmann, J. P., & Wittekindt, C. (2019) · Oral Oncology · doi:10.1016/j.oraloncology.2019.04.013 · Cited by 14 · see citing works
Millisecond molecular dynamics simulations of KRas-dimer formation and interfaces
Ngo, V. A., & Garcı́a, A. E. (2022) · Biophysical Journal · doi:10.1016/j.bpj.2022.04.026 · Cited by 13 · see citing works
Tripathi, S., Dsouza, N. R., Mathison, A., Leverence, E. N., Urrutia, R. A., & Zimmermann, M. T. (2021) · Computational and Structural Biotechnology Journal · doi:10.1016/j.csbj.2021.12.007 · Cited by 11 · see citing works
“From these patterns among hotspot variants, we inferred certain amino acid substitutions have the same mechanisms across RAS proteins, while other substitutions are modulated by intrinsic biochemical properties of individual RAS genes [38].”
BackgroundJani, V., Sonavane, U., & Joshi, R. (2024) · Heliyon · doi:10.1016/j.heliyon.2024.e36161 · Cited by 11 · see citing works
“Also, Johnson et al. has shown that the three isoforms KRAS, HRAS and NRAS are biochemically distinct [52].”
BackgroundTripathi, S., Dsouza, N. R., Urrutia, R. A., & Zimmermann, M. T. (2020) · Bioinformatics · doi:10.1093/bioinformatics/btaa972 · Cited by 11 · see citing works
How to make an undruggable enzyme druggable: lessons from ras proteins
Sayyed–Ahmad, A., & Gorfe, A. A. (2020) · Advances in protein chemistry and structural biology · doi:10.1016/bs.apcsb.2020.05.003 · Cited by 10 · see citing works
Biophysical and Structural Characterization of Novel RAS-Binding Domains (RBDs) of PI3Kα and PI3Kγ
Martinez, N., Thieker, D. F., Carey, L. M., Rasquinha, J. A., Kistler, S., Kuhlman, B. A., & Campbell, S. L. (2021) · Journal of Molecular Biology · doi:10.1016/j.jmb.2021.166838 · Cited by 10 · see citing works
“The PI3K RBDs are more structurally similar to the RALGDS-RBD [34] and bind with similar affinity to RAS relative to the CRAF-RBD [35].”
BackgroundStructures of RGL1 RAS-Association Domain in Complex with KRAS and the Oncogenic G12V Mutant
Eves, B. J., Gebregiworgis, T., Gasmi-Seabrook, G. M. C., Kuntz, D. A., Privé, G. G., Marshall, C. B., & Ikura, M. (2022) · Journal of Molecular Biology · doi:10.1016/j.jmb.2022.167527 · Cited by 10 · see citing works
Nair, V. V., Yin, G., Zhang, J., Hancock, J. F., Campbell, S. L., & Gorfe, A. A. (2021) · The Journal of Physical Chemistry B · doi:10.1021/acs.jpcb.1c01062 · Cited by 9 · see citing works
Ilter, M., Kaşmer, R., Jalalypour, F., Atılgan, C., Topcu, O., Karakaş, N., & Şensoy, Ö. (2022) · eLife · doi:10.7554/elife.79747 · Cited by 8 · see citing works
Tisi, R., Spinelli, M., Palmioli, A., Airoldi, C., Cazzaniga, P., Besozzi, D., Nobile, M. S., Mazzoleni, E., Arnhold, S., GIOIA, L. D., et al. (2021) · Frontiers in Molecular Biosciences · doi:10.3389/fmolb.2021.625979 · Cited by 8 · see citing works
“, 2019), appropriately modified according to recent data (Johnson et al., 2017; Johnson et al., 2019), predicts the ability of cmp4 to inhibit the proliferation of different Ras-driven cancer cells.”
BackgroundCrystal structure of NRAS Q61K with a ligand-induced pocket near switch II
Gebregiworgis, T., Chan, J. Y.-L., Kuntz, D. A., Privé, G. G., Marshall, C. B., & Ikura, M. (2024) · European Journal of Cell Biology · doi:10.1016/j.ejcb.2024.151414 · Cited by 9 · see citing works
Sabt, A., Tawfik, H. O., Khaleel, E. F., Badi, R. M., Ibrahim, H. A. A., Elkaeed, E. B., & Eldehna, W. M. (2024) · Molecular Diversity · doi:10.1007/s11030-023-10777-6 · Cited by 7 · see citing works
“The RAS protein family contains three known human isoforms H-RAS, N-RAS, and K-RAS [8, 9].”
Adeniji, E. A., Olotu, F. A., & Soliman, M. E. S. (2018) · Anti-Cancer Agents in Medicinal Chemistry · doi:10.2174/1871520618666180718110231 · Cited by 7 · see citing works
Predicting X‐ray solution scattering from flexible macromolecules
Zhou, H., Guterres, H., Mattos, C., & Makowski, L. (2018) · Protein Science · doi:10.1002/pro.3508 · Cited by 7 · see citing works
“lated by an allosteric site, which promotes the transition from a disordered T-state to an ordered Rstate conformation conducive to GTP hydrolysis.(25,28)”
BackgroundSelf-citationPrajapati, V., Singh, A. K., Kumar, A. M., Singh, H., Pathak, P., Grishina, M., Kumar, V., Khalilullah, H., Verma, A., & Kumar, P. (2025) · RSC Medicinal Chemistry · doi:10.1039/d4md00923a · Cited by 6 · see citing works
Khalid, A. Q., Zaidan, T. N., Bhuvanendran, S., Magalingam, K. B., Mohamedahmed, S. M., Ramdas, P., & Radhakrishnan, A. K. (2024) · Nutrition Reviews · doi:10.1093/nutrit/nuae108 · Cited by 6 · see citing works
Ras Isoforms from Lab Benches to Lives—What Are We Missing and How Far Are We?
Nair, A., Kubatzky, K. F., & Saha, B. (2021) · International Journal of Molecular Sciences · doi:10.3390/ijms22126508 · Cited by 6 · see citing works
Yu, R. T. D., & Garcia, R. L. (2020) · Scientific Reports · doi:10.1038/s41598-020-67796-8 · Cited by 6 · see citing works
Adamopoulos, P. G., Tsiakanikas, P., Boti, M. A., & Scorilas, A. (2021) · International Journal of Molecular Sciences · doi:10.3390/ijms222413298 · Cited by 6 · see citing works
“The allosteric lobe contains hot spots of protein–ligand interactions that bind membrane components and harbors all the differences between the RAS G-domains at the level of primary structure, with a sequence identity of 90% among the allosteric lobes of the three RAS proteins [11,13,14].”
BackgroundNeuroblastoma RAS viral oncogene homolog (N-RAS) deficiency aggravates liver injury and fibrosis
Zheng, K., Hao, F., Medrano-Garcia, S., Chen, C., Guo, F., Morán-Blanco, L., Rodríguez, S., Torres-Ruiz, R., Peligros, M. I., Vaquero, J., et al. (2023) · Cell Death and Disease · doi:10.1038/s41419-023-06029-y · Cited by 5 · see citing works
“All mice were sacri fi ced 28 days after BDL.”
Water in Ras Superfamily Evolution
Marcus, K., & Mattos, C. (2019) · Journal of Computational Chemistry · doi:10.1002/jcc.26060 · Cited by 5 · see citing works
Self-citationCiftci, F. S., & Erman, B. (2026) · Proteins Structure Function and Bioinformatics · doi:10.1002/prot.70146 · Cited by 4 · see citing works
Nigam, A., Krishnamoorthy, G. P., Chatila, W. K., Berman, K., Saqcena, M., Walch, H. S., Venkatramani, M., Ho, A. L., Schultz, N. D., Fagin, J. A., et al. (2024) · Oncogene · doi:10.1038/s41388-024-03095-0 · Cited by 4 · see citing works
“Characterization and transfection of “ RASless ” mouse embryonic fi broblasts”
Antipsychotic phenothiazine drugs bind to KRAS in vitro
Wang, X., Gorfe, A. A., & Putkey, J. A. (2021) · Journal of Biomolecular NMR · doi:10.1007/s10858-021-00371-z · Cited by 4 · see citing works
Girard, É., Lopes, P., Spoerner, M., Dhaussy, A., Prangé, T., Kalbitzer, H. R., & Colloc’h, N. (2024) · Chemistry - A European Journal · doi:10.1002/chem.202400304 · Cited by 4 · see citing works
Elucidating Ras protein as a dual therapeutic target for inflammation and cancer: a review
Nandan, P. K., & Jayanthi, S. (2025) · Discover Oncology · doi:10.1007/s12672-025-02783-x · Cited by 3 · see citing works
“It is believed that variable intracellular localization, which favours preferential coupling to particular effector pathways, mediates isoform-specific signalling [51–53] and by unique biochemical characteristics contributed by allosteric lobe sequence differences among each isoform [54].”
PANCS-spec-Binders: A system for rapidly discovering isoform- or epitope-specific binders
Pixley, J. A., Styles, M. J., Aphicho, K., Endres, M., Gade, P., Michalska, K., Joachimiak, A., & Dickinson, B. C. (2026) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.2531008123 · Cited by 3 · see citing works
Revealing conformational changes of GTP-bound NRAS mutants probed by GaMD and Markov state models
Zhao, L., Wang, J., Ping, H., Yang, W., Cao, H., & Chen, J. (2026) · Physical Chemistry Chemical Physics · doi:10.1039/d5cp04245c · Cited by 3 · see citing works
Knihtila, R., Volmar, A. Y., Meilleur, F., & Mattos, C. (2019) · Acta Crystallographica Section F Structural Biology Communications · doi:10.1107/s2053230x18018125 · Cited by 3 · see citing works
“Furthermore, it has become evident that GTPases are highly allosteric and that the networks across the G-domain play a critical role in the biochemistry of these proteins (Johnson et al., 2017).”
BackgroundSelf-citationGTP-Bound N-Ras Conformational States and Substates Are Modulated by Membrane and Point Mutation
Farcaș, A., & Janosi, L. (2024) · International Journal of Molecular Sciences · doi:10.3390/ijms25031430 · Cited by 3 · see citing works
“Hence, an order of magnitude fewer crystallographic structures are available for N-Ras (e.g., GDP-bound N-Ras (3CON) [51] and GTP-equivalent GppNHp-bound N-Ras (5UHV) [52]).”
BackgroundMethodProtCID: A data resource for structural information on protein interactionsPreprint
Xu, Q., & Dunbrack, R. L. (2019) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/579862 · Cited by 3 · see citing works
Post, J. B., Hami, N., Lohuis, J. O., van de Ven, M., de Korte‐Grimmerink, R., Stangl, C., Stelloo, E., Verlaan-Klink, I., van Rheenen, J., & Snippert, H. J. G. (2019) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/860122 · Cited by 3 · see citing works
- 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
- Venue
- Journal of Biological Chemistry
- Publisher
- Elsevier BV
- Published
- 2017-08
- Volume
- 292
- Issue
- 31
- Pages
- 12981-12993
- ISSN
- 0021-9258 (ISSN-L)
- 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
- 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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