Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α

Autoren

Elise Delaforge
Sigrid Milles
Guillaume Bouvignies
Denis Bouvier
Stephane Boivin
Nicola Salvi
Damien Maurin
Anne Martel
Adam Round
Edward A Lemke
Malene Ringkjøbing Jensen
Darren J Hart
Martin Blackledge

DOI

10.1021/jacs.5b07765

eISSN

1520-5126

ISSN

0002-7863

Ausgabe der Veröffentlichung

48

Zeitschrift

Journal of the American Chemical Society

Sprache

en

Online publication date

2015

Paginierung

15122 - 15134

Datum der Veröffentlichung

2015

Status

Published

Herausgeber

American Chemical Society (ACS)

Herausgeber URL

http://dx.doi.org/10.1021/jacs.5b07765

Datum der Datenerfassung

2023

Titel

Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α

Ausgabe der Zeitschrift

137

Datenquelle: Crossref

Abstract

Influenza A RNA polymerase complex is formed from three components, PA, PB1, and PB2. PB2 is independently imported into the nucleus prior to polymerase reconstitution. All crystallographic structures of the PB2 C-terminus (residues 536-759) reveal two globular domains, 627 and NLS, that form a tightly packed heterodimer. The molecular basis of the affinity of 627-NLS for importins remained unclear from these structures, apparently requiring large-scale conformational changes prior to importin binding. Using a combination of solution-state NMR, small-angle neutron scattering, small-angle X-ray scattering (SAXS), and Förster resonance energy transfer (FRET), we show that 627-NLS populates a temperature-dependent dynamic equilibrium between closed and open states. The closed state is stabilized by a tripartite salt bridge involving the 627-NLS interface and the linker, that becomes flexible in the open state, with 627 and NLS dislocating into a highly dynamic ensemble. Activation enthalpies and entropies associated with the rupture of this interface were derived from simultaneous analysis of temperature-dependent chemical exchange saturation transfer measurements, revealing a strong temperature dependence of both open-state population and exchange rate. Single-molecule FRET and SAXS demonstrate that only the open-form is capable of binding to importin α and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin α. This intrinsic large-scale conformational flexibility therefore enables 627-NLS to bind importin through conformational selection from a temperature-dependent equilibrium comprising both functional forms of the protein.

Addresses

Univ. Grenoble Alpes , Institut de Biologie Structurale (IBS), F-38044 Grenoble, France.

Autoren

Elise Delaforge
Sigrid Milles
Guillaume Bouvignies
Denis Bouvier
Stephane Boivin
Nicola Salvi
Damien Maurin
Anne Martel
Adam Round
Edward A Lemke
Malene Ringkjøbing Jensen
Darren J Hart
Martin Blackledge

DOI

10.1021/jacs.5b07765

eISSN

1520-5126

Externe Identifier

PubMed Identifier: 26424125

Funding acknowledgements

Agence Nationale de la Recherche:
R?gion Rh?ne -Alpes:
Schweizerische Nationalfonds zur F?rderung der Wissenschaftlichen Forschung: P2ELP2_148858
European Molecular Biology Organization:
FINOVI Foundation:

Open access

false

ISSN

0002-7863

Ausgabe der Veröffentlichung

48

Zeitschrift

Journal of the American Chemical Society

Schlüsselwörter

Karyopherins
Viral Proteins
Solutions
Crystallography, X-Ray
Fluorescence Resonance Energy Transfer
Nuclear Magnetic Resonance, Biomolecular
Protein Conformation
Protein Binding
Influenza A Virus, H5N1 Subtype

Sprache

eng

Medium

Print-Electronic

Online publication date

2015

Paginierung

15122 - 15134

Datum der Veröffentlichung

2015

Status

Published

Datum der Datenerfassung

2015

Titel

Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α.

Sub types

Research Support, Non-U.S. Gov't
Journal Article

Ausgabe der Zeitschrift

137

Datenquelle: Europe PubMed Central

Abstract

Influenza A RNA polymerase complex is formed from three components, PA, PB1, and PB2. PB2 is independently imported into the nucleus prior to polymerase reconstitution. All crystallographic structures of the PB2 C-terminus (residues 536-759) reveal two globular domains, 627 and NLS, that form a tightly packed heterodimer. The molecular basis of the affinity of 627-NLS for importins remained unclear from these structures, apparently requiring large-scale conformational changes prior to importin binding. Using a combination of solution-state NMR, small-angle neutron scattering, small-angle X-ray scattering (SAXS), and Förster resonance energy transfer (FRET), we show that 627-NLS populates a temperature-dependent dynamic equilibrium between closed and open states. The closed state is stabilized by a tripartite salt bridge involving the 627-NLS interface and the linker, that becomes flexible in the open state, with 627 and NLS dislocating into a highly dynamic ensemble. Activation enthalpies and entropies associated with the rupture of this interface were derived from simultaneous analysis of temperature-dependent chemical exchange saturation transfer measurements, revealing a strong temperature dependence of both open-state population and exchange rate. Single-molecule FRET and SAXS demonstrate that only the open-form is capable of binding to importin α and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin α. This intrinsic large-scale conformational flexibility therefore enables 627-NLS to bind importin through conformational selection from a temperature-dependent equilibrium comprising both functional forms of the protein.

Autoren

Elise Delaforge
Sigrid Milles
Guillaume Bouvignies
Denis Bouvier
Stephane Boivin
Nicola Salvi
Damien Maurin
Anne Martel
Adam Round
Edward A Lemke
Malene Ringkjøbing Jensen
Darren J Hart
Martin Blackledge

Autoren-URL

https://www.ncbi.nlm.nih.gov/pubmed/26424125

DOI

10.1021/jacs.5b07765

eISSN

1520-5126

Ausgabe der Veröffentlichung

48

Zeitschrift

J Am Chem Soc

Schlüsselwörter

Crystallography, X-Ray
Fluorescence Resonance Energy Transfer
Influenza A Virus, H5N1 Subtype
Karyopherins
Nuclear Magnetic Resonance, Biomolecular
Protein Binding
Protein Conformation
Solutions
Viral Proteins

Sprache

eng

Country

United States

Paginierung

15122 - 15134

Datum der Veröffentlichung

2015

Status

Published

Datum, an dem der Datensatz öffentlich gemacht wurde

2016

Titel

Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α.

Sub types

Journal Article
Research Support, Non-U.S. Gov't

Ausgabe der Zeitschrift

137

Datenquelle: PubMed

Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α

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