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Allosteric inactivation of an engineered optogenetic GTPase

Optogenetics is a technique for establishing direct spatiotemporal control over molecular function within living cells using light. Light application induces conformational changes within targeted proteins that produce changes in function. One of the applications of optogenetic tools is an allosteri...

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Autores principales: Jain, Abha, Dokholyan, Nikolay V., Lee, Andrew L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10083549/
https://www.ncbi.nlm.nih.gov/pubmed/36972433
http://dx.doi.org/10.1073/pnas.2219254120
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author Jain, Abha
Dokholyan, Nikolay V.
Lee, Andrew L.
author_facet Jain, Abha
Dokholyan, Nikolay V.
Lee, Andrew L.
author_sort Jain, Abha
collection PubMed
description Optogenetics is a technique for establishing direct spatiotemporal control over molecular function within living cells using light. Light application induces conformational changes within targeted proteins that produce changes in function. One of the applications of optogenetic tools is an allosteric control of proteins via light-sensing domain (LOV2), which allows direct and robust control of protein function. Computational studies supported by cellular imaging demonstrated that application of light allosterically inhibited signaling proteins Vav2, ITSN, and Rac1, but the structural and dynamic basis of such control has yet to be elucidated by experiment. Here, using NMR spectroscopy, we discover principles of action of allosteric control of cell division control protein 42 (CDC42), a small GTPase involved in cell signaling. Both LOV2 and Cdc42 employ flexibility in their function to switch between “dark”/“lit” or active/inactive states, respectively. By conjoining Cdc42 and phototropin1 LOV2 domains into the bi-switchable fusion Cdc42Lov, application of light—or alternatively, mutation in LOV2 to mimic light absorption—allosterically inhibits Cdc42 downstream signaling. The flow and patterning of allosteric transduction in this flexible system are well suited to observation by NMR. Close monitoring of the structural and dynamic properties of dark versus “lit” states of Cdc42Lov revealed lit-induced allosteric perturbations that extend to Cdc42’s downstream effector binding site. Chemical shift perturbations for lit mimic, I539E, have distinct regions of sensitivity, and both the domains are coupled together, leading to bidirectional interdomain signaling. Insights gained from this optoallosteric design will increase our ability to control response sensitivity in future designs.
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spelling pubmed-100835492023-09-27 Allosteric inactivation of an engineered optogenetic GTPase Jain, Abha Dokholyan, Nikolay V. Lee, Andrew L. Proc Natl Acad Sci U S A Biological Sciences Optogenetics is a technique for establishing direct spatiotemporal control over molecular function within living cells using light. Light application induces conformational changes within targeted proteins that produce changes in function. One of the applications of optogenetic tools is an allosteric control of proteins via light-sensing domain (LOV2), which allows direct and robust control of protein function. Computational studies supported by cellular imaging demonstrated that application of light allosterically inhibited signaling proteins Vav2, ITSN, and Rac1, but the structural and dynamic basis of such control has yet to be elucidated by experiment. Here, using NMR spectroscopy, we discover principles of action of allosteric control of cell division control protein 42 (CDC42), a small GTPase involved in cell signaling. Both LOV2 and Cdc42 employ flexibility in their function to switch between “dark”/“lit” or active/inactive states, respectively. By conjoining Cdc42 and phototropin1 LOV2 domains into the bi-switchable fusion Cdc42Lov, application of light—or alternatively, mutation in LOV2 to mimic light absorption—allosterically inhibits Cdc42 downstream signaling. The flow and patterning of allosteric transduction in this flexible system are well suited to observation by NMR. Close monitoring of the structural and dynamic properties of dark versus “lit” states of Cdc42Lov revealed lit-induced allosteric perturbations that extend to Cdc42’s downstream effector binding site. Chemical shift perturbations for lit mimic, I539E, have distinct regions of sensitivity, and both the domains are coupled together, leading to bidirectional interdomain signaling. Insights gained from this optoallosteric design will increase our ability to control response sensitivity in future designs. National Academy of Sciences 2023-03-27 2023-04-04 /pmc/articles/PMC10083549/ /pubmed/36972433 http://dx.doi.org/10.1073/pnas.2219254120 Text en Copyright © 2023 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by-nc-nd/4.0/This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/) .
spellingShingle Biological Sciences
Jain, Abha
Dokholyan, Nikolay V.
Lee, Andrew L.
Allosteric inactivation of an engineered optogenetic GTPase
title Allosteric inactivation of an engineered optogenetic GTPase
title_full Allosteric inactivation of an engineered optogenetic GTPase
title_fullStr Allosteric inactivation of an engineered optogenetic GTPase
title_full_unstemmed Allosteric inactivation of an engineered optogenetic GTPase
title_short Allosteric inactivation of an engineered optogenetic GTPase
title_sort allosteric inactivation of an engineered optogenetic gtpase
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10083549/
https://www.ncbi.nlm.nih.gov/pubmed/36972433
http://dx.doi.org/10.1073/pnas.2219254120
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