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A saturation-mutagenesis analysis of the interplay between stability and activation in Ras

Cancer mutations in Ras occur predominantly at three hotspots: Gly 12, Gly 13, and Gln 61. Previously, we reported that deep mutagenesis of H-Ras using a bacterial assay identified many other activating mutations (Bandaru et al., 2017). We now show that the results of saturation mutagenesis of H-Ras...

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Autores principales: Hidalgo, Frank, Nocka, Laura M, Shah, Neel H, Gorday, Kent, Latorraca, Naomi R, Bandaru, Pradeep, Templeton, Sage, Lee, David, Karandur, Deepti, Pelton, Jeffrey G, Marqusee, Susan, Wemmer, David, Kuriyan, John
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8916776/
https://www.ncbi.nlm.nih.gov/pubmed/35272765
http://dx.doi.org/10.7554/eLife.76595
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author Hidalgo, Frank
Nocka, Laura M
Shah, Neel H
Gorday, Kent
Latorraca, Naomi R
Bandaru, Pradeep
Templeton, Sage
Lee, David
Karandur, Deepti
Pelton, Jeffrey G
Marqusee, Susan
Wemmer, David
Kuriyan, John
author_facet Hidalgo, Frank
Nocka, Laura M
Shah, Neel H
Gorday, Kent
Latorraca, Naomi R
Bandaru, Pradeep
Templeton, Sage
Lee, David
Karandur, Deepti
Pelton, Jeffrey G
Marqusee, Susan
Wemmer, David
Kuriyan, John
author_sort Hidalgo, Frank
collection PubMed
description Cancer mutations in Ras occur predominantly at three hotspots: Gly 12, Gly 13, and Gln 61. Previously, we reported that deep mutagenesis of H-Ras using a bacterial assay identified many other activating mutations (Bandaru et al., 2017). We now show that the results of saturation mutagenesis of H-Ras in mammalian Ba/F3 cells correlate well with the results of bacterial experiments in which H-Ras or K-Ras are co-expressed with a GTPase-activating protein (GAP). The prominent cancer hotspots are not dominant in the Ba/F3 data. We used the bacterial system to mutagenize Ras constructs of different stabilities and discovered a feature that distinguishes the cancer hotspots. While mutations at the cancer hotspots activate Ras regardless of construct stability, mutations at lower-frequency sites (e.g. at Val 14 or Asp 119) can be activating or deleterious, depending on the stability of the Ras construct. We characterized the dynamics of three non-hotspot activating Ras mutants by using NMR to monitor hydrogen-deuterium exchange (HDX). These mutations result in global increases in HDX rates, consistent with destabilization of Ras. An explanation for these observations is that mutations that destabilize Ras increase nucleotide dissociation rates, enabling activation by spontaneous nucleotide exchange. A further stability decrease can lead to insufficient levels of folded Ras – and subsequent loss of function. In contrast, the cancer hotspot mutations are mechanism-based activators of Ras that interfere directly with the action of GAPs. Our results demonstrate the importance of GAP surveillance and protein stability in determining the sensitivity of Ras to mutational activation.
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spelling pubmed-89167762022-03-12 A saturation-mutagenesis analysis of the interplay between stability and activation in Ras Hidalgo, Frank Nocka, Laura M Shah, Neel H Gorday, Kent Latorraca, Naomi R Bandaru, Pradeep Templeton, Sage Lee, David Karandur, Deepti Pelton, Jeffrey G Marqusee, Susan Wemmer, David Kuriyan, John eLife Structural Biology and Molecular Biophysics Cancer mutations in Ras occur predominantly at three hotspots: Gly 12, Gly 13, and Gln 61. Previously, we reported that deep mutagenesis of H-Ras using a bacterial assay identified many other activating mutations (Bandaru et al., 2017). We now show that the results of saturation mutagenesis of H-Ras in mammalian Ba/F3 cells correlate well with the results of bacterial experiments in which H-Ras or K-Ras are co-expressed with a GTPase-activating protein (GAP). The prominent cancer hotspots are not dominant in the Ba/F3 data. We used the bacterial system to mutagenize Ras constructs of different stabilities and discovered a feature that distinguishes the cancer hotspots. While mutations at the cancer hotspots activate Ras regardless of construct stability, mutations at lower-frequency sites (e.g. at Val 14 or Asp 119) can be activating or deleterious, depending on the stability of the Ras construct. We characterized the dynamics of three non-hotspot activating Ras mutants by using NMR to monitor hydrogen-deuterium exchange (HDX). These mutations result in global increases in HDX rates, consistent with destabilization of Ras. An explanation for these observations is that mutations that destabilize Ras increase nucleotide dissociation rates, enabling activation by spontaneous nucleotide exchange. A further stability decrease can lead to insufficient levels of folded Ras – and subsequent loss of function. In contrast, the cancer hotspot mutations are mechanism-based activators of Ras that interfere directly with the action of GAPs. Our results demonstrate the importance of GAP surveillance and protein stability in determining the sensitivity of Ras to mutational activation. eLife Sciences Publications, Ltd 2022-03-11 /pmc/articles/PMC8916776/ /pubmed/35272765 http://dx.doi.org/10.7554/eLife.76595 Text en © 2022, Hidalgo et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Structural Biology and Molecular Biophysics
Hidalgo, Frank
Nocka, Laura M
Shah, Neel H
Gorday, Kent
Latorraca, Naomi R
Bandaru, Pradeep
Templeton, Sage
Lee, David
Karandur, Deepti
Pelton, Jeffrey G
Marqusee, Susan
Wemmer, David
Kuriyan, John
A saturation-mutagenesis analysis of the interplay between stability and activation in Ras
title A saturation-mutagenesis analysis of the interplay between stability and activation in Ras
title_full A saturation-mutagenesis analysis of the interplay between stability and activation in Ras
title_fullStr A saturation-mutagenesis analysis of the interplay between stability and activation in Ras
title_full_unstemmed A saturation-mutagenesis analysis of the interplay between stability and activation in Ras
title_short A saturation-mutagenesis analysis of the interplay between stability and activation in Ras
title_sort saturation-mutagenesis analysis of the interplay between stability and activation in ras
topic Structural Biology and Molecular Biophysics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8916776/
https://www.ncbi.nlm.nih.gov/pubmed/35272765
http://dx.doi.org/10.7554/eLife.76595
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