Structural and regulatory elements of HCV NS5B polymerase--β-loop and C-terminal tail--are required for activity of allosteric thumb site II inhibitors.

Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well underst...

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Autores principales:	Sarah E Boyce, Neeraj Tirunagari, Anita Niedziela-Majka, Jason Perry, Melanie Wong, Elaine Kan, Leanna Lagpacan, Ona Barauskas, Magdeleine Hung, Martijn Fenaux, Todd Appleby, William J Watkins, Uli Schmitz, Roman Sakowicz
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/511db7137be1485baa683530d590daed
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spelling	oai:doaj.org-article:511db7137be1485baa683530d590daed2021-11-18T08:38:12ZStructural and regulatory elements of HCV NS5B polymerase--β-loop and C-terminal tail--are required for activity of allosteric thumb site II inhibitors.1932-620310.1371/journal.pone.0084808https://doaj.org/article/511db7137be1485baa683530d590daed2014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24416288/?tool=EBIhttps://doaj.org/toc/1932-6203Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B--the C-terminal tail and β-loop--in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the β-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor-NS5B complex are absent in the inhibitor-bound constructs in which interactions between C-terminal tail and β-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and β-loop.Sarah E BoyceNeeraj TirunagariAnita Niedziela-MajkaJason PerryMelanie WongElaine KanLeanna LagpacanOna BarauskasMagdeleine HungMartijn FenauxTodd ApplebyWilliam J WatkinsUli SchmitzRoman SakowiczPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 1, p e84808 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Sarah E Boyce Neeraj Tirunagari Anita Niedziela-Majka Jason Perry Melanie Wong Elaine Kan Leanna Lagpacan Ona Barauskas Magdeleine Hung Martijn Fenaux Todd Appleby William J Watkins Uli Schmitz Roman Sakowicz Structural and regulatory elements of HCV NS5B polymerase--β-loop and C-terminal tail--are required for activity of allosteric thumb site II inhibitors.
description	Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B--the C-terminal tail and β-loop--in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the β-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor-NS5B complex are absent in the inhibitor-bound constructs in which interactions between C-terminal tail and β-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and β-loop.
format	article
author	Sarah E Boyce Neeraj Tirunagari Anita Niedziela-Majka Jason Perry Melanie Wong Elaine Kan Leanna Lagpacan Ona Barauskas Magdeleine Hung Martijn Fenaux Todd Appleby William J Watkins Uli Schmitz Roman Sakowicz
author_facet	Sarah E Boyce Neeraj Tirunagari Anita Niedziela-Majka Jason Perry Melanie Wong Elaine Kan Leanna Lagpacan Ona Barauskas Magdeleine Hung Martijn Fenaux Todd Appleby William J Watkins Uli Schmitz Roman Sakowicz
author_sort	Sarah E Boyce
title	Structural and regulatory elements of HCV NS5B polymerase--β-loop and C-terminal tail--are required for activity of allosteric thumb site II inhibitors.
title_short	Structural and regulatory elements of HCV NS5B polymerase--β-loop and C-terminal tail--are required for activity of allosteric thumb site II inhibitors.
title_full	Structural and regulatory elements of HCV NS5B polymerase--β-loop and C-terminal tail--are required for activity of allosteric thumb site II inhibitors.
title_fullStr	Structural and regulatory elements of HCV NS5B polymerase--β-loop and C-terminal tail--are required for activity of allosteric thumb site II inhibitors.
title_full_unstemmed	Structural and regulatory elements of HCV NS5B polymerase--β-loop and C-terminal tail--are required for activity of allosteric thumb site II inhibitors.
title_sort	structural and regulatory elements of hcv ns5b polymerase--β-loop and c-terminal tail--are required for activity of allosteric thumb site ii inhibitors.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/511db7137be1485baa683530d590daed
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Structural and regulatory elements of HCV NS5B polymerase--β-loop and C-terminal tail--are required for activity of allosteric thumb site II inhibitors.

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