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Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer

[Image: see text] Ribonucleotide reductases (RNRs) are responsible for all de novo biosynthesis of DNA precursors in nature by catalyzing the conversion of ribonucleotides to deoxyribonucleotides. Because of its essential role in cell division, human RNR is a target for a number of anticancer drugs...

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Autores principales: Ando, Nozomi, Li, Haoran, Brignole, Edward J., Thompson, Samuel, McLaughlin, Martin I., Page, Julia E., Asturias, Francisco J., Stubbe, JoAnne, Drennan, Catherine L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2015
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4722859/
https://www.ncbi.nlm.nih.gov/pubmed/26727048
http://dx.doi.org/10.1021/acs.biochem.5b01207
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author Ando, Nozomi
Li, Haoran
Brignole, Edward J.
Thompson, Samuel
McLaughlin, Martin I.
Page, Julia E.
Asturias, Francisco J.
Stubbe, JoAnne
Drennan, Catherine L.
author_facet Ando, Nozomi
Li, Haoran
Brignole, Edward J.
Thompson, Samuel
McLaughlin, Martin I.
Page, Julia E.
Asturias, Francisco J.
Stubbe, JoAnne
Drennan, Catherine L.
author_sort Ando, Nozomi
collection PubMed
description [Image: see text] Ribonucleotide reductases (RNRs) are responsible for all de novo biosynthesis of DNA precursors in nature by catalyzing the conversion of ribonucleotides to deoxyribonucleotides. Because of its essential role in cell division, human RNR is a target for a number of anticancer drugs in clinical use. Like other class Ia RNRs, human RNR requires both a radical-generation subunit (β) and nucleotide-binding subunit (α) for activity. Because of their complex dependence on allosteric effectors, however, the active and inactive quaternary forms of many class Ia RNRs have remained in question. Here, we present an X-ray crystal structure of the human α subunit in the presence of inhibiting levels of dATP, depicting a ring-shaped hexamer (α(6)) where the active sites line the inner hole. Surprisingly, our small-angle X-ray scattering (SAXS) results indicate that human α forms a similar hexamer in the presence of ATP, an activating effector. In both cases, α(6) is assembled from dimers (α(2)) without a previously proposed tetramer intermediate (α(4)). However, we show with SAXS and electron microscopy that at millimolar ATP, the ATP-induced α(6) can further interconvert with higher-order filaments. Differences in the dATP- and ATP-induced α(6) were further examined by SAXS in the presence of the β subunit and by activity assays as a function of ATP or dATP. Together, these results suggest that dATP-induced α(6) is more stable than the ATP-induced α(6) and that stabilization of this ring-shaped configuration provides a mechanism to prevent access of the β subunit to the active site of α.
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spelling pubmed-47228592016-12-21 Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer Ando, Nozomi Li, Haoran Brignole, Edward J. Thompson, Samuel McLaughlin, Martin I. Page, Julia E. Asturias, Francisco J. Stubbe, JoAnne Drennan, Catherine L. Biochemistry [Image: see text] Ribonucleotide reductases (RNRs) are responsible for all de novo biosynthesis of DNA precursors in nature by catalyzing the conversion of ribonucleotides to deoxyribonucleotides. Because of its essential role in cell division, human RNR is a target for a number of anticancer drugs in clinical use. Like other class Ia RNRs, human RNR requires both a radical-generation subunit (β) and nucleotide-binding subunit (α) for activity. Because of their complex dependence on allosteric effectors, however, the active and inactive quaternary forms of many class Ia RNRs have remained in question. Here, we present an X-ray crystal structure of the human α subunit in the presence of inhibiting levels of dATP, depicting a ring-shaped hexamer (α(6)) where the active sites line the inner hole. Surprisingly, our small-angle X-ray scattering (SAXS) results indicate that human α forms a similar hexamer in the presence of ATP, an activating effector. In both cases, α(6) is assembled from dimers (α(2)) without a previously proposed tetramer intermediate (α(4)). However, we show with SAXS and electron microscopy that at millimolar ATP, the ATP-induced α(6) can further interconvert with higher-order filaments. Differences in the dATP- and ATP-induced α(6) were further examined by SAXS in the presence of the β subunit and by activity assays as a function of ATP or dATP. Together, these results suggest that dATP-induced α(6) is more stable than the ATP-induced α(6) and that stabilization of this ring-shaped configuration provides a mechanism to prevent access of the β subunit to the active site of α. American Chemical Society 2015-12-21 2016-01-19 /pmc/articles/PMC4722859/ /pubmed/26727048 http://dx.doi.org/10.1021/acs.biochem.5b01207 Text en Copyright © 2015 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes.
spellingShingle Ando, Nozomi
Li, Haoran
Brignole, Edward J.
Thompson, Samuel
McLaughlin, Martin I.
Page, Julia E.
Asturias, Francisco J.
Stubbe, JoAnne
Drennan, Catherine L.
Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer
title Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer
title_full Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer
title_fullStr Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer
title_full_unstemmed Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer
title_short Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer
title_sort allosteric inhibition of human ribonucleotide reductase by datp entails the stabilization of a hexamer
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4722859/
https://www.ncbi.nlm.nih.gov/pubmed/26727048
http://dx.doi.org/10.1021/acs.biochem.5b01207
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