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

Delaforge, Elise, Milles, Sigrid, Bouvignies, Guillaume, Bouvier, Denis, Boivin, Stephane, Salvi, Nicola, Maurin, Damien, Martel, Anne, Round, Adam, Lemke, Edward A., Ringkjøbing Jensen, Malene, Hart, Darren J., Blackledge, Martin
Journal of the American Chemical Society 2015 v.137 no.48 pp. 15122-15134
DNA-directed RNA polymerase, X-radiation, energy transfer, enthalpy, importins, influenza, nuclear magnetic resonance spectroscopy
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.