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J Am Chem Soc. 2015 Dec 9;137(48):15122-34. doi: 10.1021/jacs.5b07765. Epub 2015 Oct 16.

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

Delaforge E1,2,3, Milles S1,2,3, Bouvignies G1,2,3, Bouvier D1,2,3,4,5,6, Boivin S4,5,6, Salvi N1,2,3, Maurin D1,2,3, Martel A7, Round A8, Lemke EA9, Jensen MR1,2,3, Hart DJ4,5,6, Blackledge M1,2,3.

Author information

1
Univ. Grenoble Alpes , Institut de Biologie Structurale (IBS), F-38044 Grenoble, France.
2
CEA, DSV, IBS , F-38044 Grenoble, France.
3
CNRS, IBS , F-38044 Grenoble, France.
4
Univ. Grenoble Alpes, UVHCI , Grenoble, France.
5
CNRS, UVHCI , Grenoble, France.
6
Unit for Virus Host Cell Interactions, Univ. Grenoble Alpes-EMBL-CNRS , Grenoble, France.
7
Institut Laue-Langevin , F-38044 Grenoble, France.
8
European Molecular Biology Laboratory , Grenoble Outstation, 38042 Grenoble, France.
9
European Molecular Biology Laboratory, Structural and Computational Biology Unit , Meyerhofstrasse 1, 69117 Heidelberg, Germany.

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.

PMID:
26424125
DOI:
10.1021/jacs.5b07765
[Indexed for MEDLINE]

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