title: Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α.
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.
authors.affiliation: Univ. Grenoble Alpes , Institut de Biologie Structurale (IBS), F-38044 Grenoble, France.|CEA, DSV, IBS , F-38044 Grenoble, France.|CNRS, IBS , F-38044 Grenoble, France.
authors.forename: Elise
authors.initials: E
authors.lastname: Delaforge
authors.affiliation: Univ. Grenoble Alpes , Institut de Biologie Structurale (IBS), F-38044 Grenoble, France.|CEA, DSV, IBS , F-38044 Grenoble, France.|CNRS, IBS , F-38044 Grenoble, France.
authors.forename: Sigrid
authors.initials: S
authors.lastname: Milles
authors.affiliation: Univ. Grenoble Alpes , Institut de Biologie Structurale (IBS), F-38044 Grenoble, France.|CEA, DSV, IBS , F-38044 Grenoble, France.|CNRS, IBS , F-38044 Grenoble, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Bouvignies
authors.affiliation: Univ. Grenoble Alpes , Institut de Biologie Structurale (IBS), F-38044 Grenoble, France.|CEA, DSV, IBS , F-38044 Grenoble, France.|CNRS, IBS , F-38044 Grenoble, France.|Univ. Grenoble Alpes, UVHCI , Grenoble, France.|CNRS, UVHCI , Grenoble, France.|Unit for Virus Host Cell Interactions, Univ. Grenoble Alpes-EMBL-CNRS , Grenoble, France.
authors.forename: Denis
authors.initials: D
authors.lastname: Bouvier
authors.affiliation: Univ. Grenoble Alpes, UVHCI , Grenoble, France.|CNRS, UVHCI , Grenoble, France.|Unit for Virus Host Cell Interactions, Univ. Grenoble Alpes-EMBL-CNRS , Grenoble, France.
authors.forename: Stephane
authors.initials: S
authors.lastname: Boivin
authors.affiliation: Univ. Grenoble Alpes , Institut de Biologie Structurale (IBS), F-38044 Grenoble, France.|CEA, DSV, IBS , F-38044 Grenoble, France.|CNRS, IBS , F-38044 Grenoble, France.
authors.forename: Nicola
authors.initials: N
authors.lastname: Salvi
authors.affiliation: Univ. Grenoble Alpes , Institut de Biologie Structurale (IBS), F-38044 Grenoble, France.|CEA, DSV, IBS , F-38044 Grenoble, France.|CNRS, IBS , F-38044 Grenoble, France.
authors.forename: Damien
authors.initials: D
authors.lastname: Maurin
authors.affiliation: Institut Laue-Langevin , F-38044 Grenoble, France.
authors.forename: Anne
authors.initials: A
authors.lastname: Martel
authors.affiliation: European Molecular Biology Laboratory , Grenoble Outstation, 38042 Grenoble, France.
authors.forename: Adam
authors.initials: A
authors.lastname: Round
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit , Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Lemke
authors.affiliation: Univ. Grenoble Alpes , Institut de Biologie Structurale (IBS), F-38044 Grenoble, France.|CEA, DSV, IBS , F-38044 Grenoble, France.|CNRS, IBS , F-38044 Grenoble, France.
authors.forename: Malene Ringkjøbing
authors.initials: MR
authors.lastname: Jensen
authors.affiliation: Univ. Grenoble Alpes, UVHCI , Grenoble, France.|CNRS, UVHCI , Grenoble, France.|Unit for Virus Host Cell Interactions, Univ. Grenoble Alpes-EMBL-CNRS , Grenoble, France.
authors.forename: Darren J
authors.initials: DJ
authors.lastname: Hart
authors.affiliation: Univ. Grenoble Alpes , Institut de Biologie Structurale (IBS), F-38044 Grenoble, France.|CEA, DSV, IBS , F-38044 Grenoble, France.|CNRS, IBS , F-38044 Grenoble, France.
authors.forename: Martin
authors.initials: M
authors.lastname: Blackledge
mesh_terms: D018360:Crystallography, X-Ray; D031541:Fluorescence Resonance Energy Transfer; D053124:Influenza A Virus, H5N1 Subtype / Q000201:enzymology*; D028884:Karyopherins / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D012996:Solutions; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D028884:Karyopherins; C502893:PB2 protein, influenza virus; D012996:Solutions; D014764:Viral Proteins