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Dynamic allostery in substrate binding by human thymidylate synthase

Human thymidylate synthase (hTS) is essential for DNA replication and therefore a therapeutic target for cancer. Effective targeting requires knowledge of the mechanism(s) of regulation of this 72 kDa homodimeric enzyme. Here, we investigate the mechanism of binding cooperativity of the nucleotide s...

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Autores principales: Bonin, Jeffrey P, Sapienza, Paul J, Lee, Andrew L
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9536839/
https://www.ncbi.nlm.nih.gov/pubmed/36200982
http://dx.doi.org/10.7554/eLife.79915
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author Bonin, Jeffrey P
Sapienza, Paul J
Lee, Andrew L
author_facet Bonin, Jeffrey P
Sapienza, Paul J
Lee, Andrew L
author_sort Bonin, Jeffrey P
collection PubMed
description Human thymidylate synthase (hTS) is essential for DNA replication and therefore a therapeutic target for cancer. Effective targeting requires knowledge of the mechanism(s) of regulation of this 72 kDa homodimeric enzyme. Here, we investigate the mechanism of binding cooperativity of the nucleotide substrate. We have employed exquisitely sensitive methyl-based CPMG and CEST NMR experiments enabling us to identify residues undergoing bifurcated linear 3-state exchange, including concerted switching between active and inactive conformations in the apo enzyme. The inactive state is populated to only ~1.3%, indicating that conformational selection contributes negligibly to the cooperativity. Instead, methyl rotation axis order parameters, determined by (2)H transverse relaxation rates, suggest that rigidification of the enzyme upon substrate binding is responsible for the entropically-driven cooperativity. Lack of the rigidification in product binding and substrate binding to an N-terminally truncated enzyme, both non-cooperative, support this idea. In addition, the lack of this rigidification in the N-terminal truncation indicates that interactions between the flexible N-terminus and the rest of the protein, which are perturbed by substrate binding, play a significant role in the cooperativity—a novel mechanism of dynamic allostery. Together, these findings yield a rare depth of insight into the substrate binding cooperativity of an essential enzyme.
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spelling pubmed-95368392022-10-07 Dynamic allostery in substrate binding by human thymidylate synthase Bonin, Jeffrey P Sapienza, Paul J Lee, Andrew L eLife Structural Biology and Molecular Biophysics Human thymidylate synthase (hTS) is essential for DNA replication and therefore a therapeutic target for cancer. Effective targeting requires knowledge of the mechanism(s) of regulation of this 72 kDa homodimeric enzyme. Here, we investigate the mechanism of binding cooperativity of the nucleotide substrate. We have employed exquisitely sensitive methyl-based CPMG and CEST NMR experiments enabling us to identify residues undergoing bifurcated linear 3-state exchange, including concerted switching between active and inactive conformations in the apo enzyme. The inactive state is populated to only ~1.3%, indicating that conformational selection contributes negligibly to the cooperativity. Instead, methyl rotation axis order parameters, determined by (2)H transverse relaxation rates, suggest that rigidification of the enzyme upon substrate binding is responsible for the entropically-driven cooperativity. Lack of the rigidification in product binding and substrate binding to an N-terminally truncated enzyme, both non-cooperative, support this idea. In addition, the lack of this rigidification in the N-terminal truncation indicates that interactions between the flexible N-terminus and the rest of the protein, which are perturbed by substrate binding, play a significant role in the cooperativity—a novel mechanism of dynamic allostery. Together, these findings yield a rare depth of insight into the substrate binding cooperativity of an essential enzyme. eLife Sciences Publications, Ltd 2022-10-06 /pmc/articles/PMC9536839/ /pubmed/36200982 http://dx.doi.org/10.7554/eLife.79915 Text en © 2022, Bonin 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
Bonin, Jeffrey P
Sapienza, Paul J
Lee, Andrew L
Dynamic allostery in substrate binding by human thymidylate synthase
title Dynamic allostery in substrate binding by human thymidylate synthase
title_full Dynamic allostery in substrate binding by human thymidylate synthase
title_fullStr Dynamic allostery in substrate binding by human thymidylate synthase
title_full_unstemmed Dynamic allostery in substrate binding by human thymidylate synthase
title_short Dynamic allostery in substrate binding by human thymidylate synthase
title_sort dynamic allostery in substrate binding by human thymidylate synthase
topic Structural Biology and Molecular Biophysics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9536839/
https://www.ncbi.nlm.nih.gov/pubmed/36200982
http://dx.doi.org/10.7554/eLife.79915
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