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: 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
• Journal: Journal of the American Chemical Society (J Am Chem Soc) Vol. 137 Issue 48 Pg. 15122-34 (Dec 09 2015) ISSN: 1520-5126 [Electronic] United States
• PMID: 26424125 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Karyopherins
• PB2 protein, influenza virus
• Solutions
• Viral Proteins

Topics

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