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Influence of Point Mutations on PR65 Conformational Adaptability: Insights from Nanoaperture Optical Tweezer Experiments and Molecular Simulations

PR65 is the HEAT-repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem-repeat protein, forming a spring-like architecture. PR65 conformational mechanics play a crucial role in PP2A function by opening/closing the substrate-binding/catalysis interface. U...

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Autores principales: Bahar, Ivet, Banerjee, Anupam, Mathew, Samuel, Naqvi, Mohsin, Yilmaz, Sema, Zachoropoulou, Maria, Doruker, Pemra, Kumita, Janet, Yang, Shang-Hua, Gur, Mert, Itzhaki, Laura, Gordon, Reuven
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Journal Experts 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10680943/
https://www.ncbi.nlm.nih.gov/pubmed/38014259
http://dx.doi.org/10.21203/rs.3.rs-3599809/v1
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author Bahar, Ivet
Banerjee, Anupam
Mathew, Samuel
Naqvi, Mohsin
Yilmaz, Sema
Zachoropoulou, Maria
Doruker, Pemra
Kumita, Janet
Yang, Shang-Hua
Gur, Mert
Itzhaki, Laura
Gordon, Reuven
author_facet Bahar, Ivet
Banerjee, Anupam
Mathew, Samuel
Naqvi, Mohsin
Yilmaz, Sema
Zachoropoulou, Maria
Doruker, Pemra
Kumita, Janet
Yang, Shang-Hua
Gur, Mert
Itzhaki, Laura
Gordon, Reuven
author_sort Bahar, Ivet
collection PubMed
description PR65 is the HEAT-repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem-repeat protein, forming a spring-like architecture. PR65 conformational mechanics play a crucial role in PP2A function by opening/closing the substrate-binding/catalysis interface. Using in-silico saturation mutagenesis we identified “hinge” residues of PR65, whose substitutions are predicted to restrict its conformational adaptability and thereby disrupt PP2A function. Molecular simulations revealed that a subset of hinge mutations stabilized the extended/open conformation, whereas another had the opposite effect. By trapping in nanoaperture optical tweezer, we characterized PR65 motion and showed that the former mutants exhibited higher corner frequencies and lower translational scattering, indicating a shift towards extended conformations, whereas the latter showed the opposite behavior. Thus, experiments confirm the conformations predicted computationally. The study highlights the utility of nanoaperture-based tweezers for exploring structure and dynamics, and the power of integrating this single-molecule method with in silico approaches.
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spelling pubmed-106809432023-11-27 Influence of Point Mutations on PR65 Conformational Adaptability: Insights from Nanoaperture Optical Tweezer Experiments and Molecular Simulations Bahar, Ivet Banerjee, Anupam Mathew, Samuel Naqvi, Mohsin Yilmaz, Sema Zachoropoulou, Maria Doruker, Pemra Kumita, Janet Yang, Shang-Hua Gur, Mert Itzhaki, Laura Gordon, Reuven Res Sq Article PR65 is the HEAT-repeat scaffold subunit of the heterotrimeric protein phosphatase 2A (PP2A) and an archetypal tandem-repeat protein, forming a spring-like architecture. PR65 conformational mechanics play a crucial role in PP2A function by opening/closing the substrate-binding/catalysis interface. Using in-silico saturation mutagenesis we identified “hinge” residues of PR65, whose substitutions are predicted to restrict its conformational adaptability and thereby disrupt PP2A function. Molecular simulations revealed that a subset of hinge mutations stabilized the extended/open conformation, whereas another had the opposite effect. By trapping in nanoaperture optical tweezer, we characterized PR65 motion and showed that the former mutants exhibited higher corner frequencies and lower translational scattering, indicating a shift towards extended conformations, whereas the latter showed the opposite behavior. Thus, experiments confirm the conformations predicted computationally. The study highlights the utility of nanoaperture-based tweezers for exploring structure and dynamics, and the power of integrating this single-molecule method with in silico approaches. American Journal Experts 2023-11-16 /pmc/articles/PMC10680943/ /pubmed/38014259 http://dx.doi.org/10.21203/rs.3.rs-3599809/v1 Text en https://creativecommons.org/licenses/by/4.0/This work is licensed under a Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/) , which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use.
spellingShingle Article
Bahar, Ivet
Banerjee, Anupam
Mathew, Samuel
Naqvi, Mohsin
Yilmaz, Sema
Zachoropoulou, Maria
Doruker, Pemra
Kumita, Janet
Yang, Shang-Hua
Gur, Mert
Itzhaki, Laura
Gordon, Reuven
Influence of Point Mutations on PR65 Conformational Adaptability: Insights from Nanoaperture Optical Tweezer Experiments and Molecular Simulations
title Influence of Point Mutations on PR65 Conformational Adaptability: Insights from Nanoaperture Optical Tweezer Experiments and Molecular Simulations
title_full Influence of Point Mutations on PR65 Conformational Adaptability: Insights from Nanoaperture Optical Tweezer Experiments and Molecular Simulations
title_fullStr Influence of Point Mutations on PR65 Conformational Adaptability: Insights from Nanoaperture Optical Tweezer Experiments and Molecular Simulations
title_full_unstemmed Influence of Point Mutations on PR65 Conformational Adaptability: Insights from Nanoaperture Optical Tweezer Experiments and Molecular Simulations
title_short Influence of Point Mutations on PR65 Conformational Adaptability: Insights from Nanoaperture Optical Tweezer Experiments and Molecular Simulations
title_sort influence of point mutations on pr65 conformational adaptability: insights from nanoaperture optical tweezer experiments and molecular simulations
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10680943/
https://www.ncbi.nlm.nih.gov/pubmed/38014259
http://dx.doi.org/10.21203/rs.3.rs-3599809/v1
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