Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin 伪
详细信息 查看全文
- 作者：Elise Delaforge ; Sigrid Milles ; Guillaume Bouvignies ; Denis Bouvier ; Stephane Boivin ; Nicola Salvi ; Damien Maurin ; Anne Martel ; Adam Round ; Edward A. Lemke ; Malene Ringkj酶bing Jensen ; Darren J. Hart ; Martin Blackledge
- 刊名：Journal of the American Chemical Society
- 出版年：2015
- 出版时间：December 9, 2015
- 年：2015
- 卷：137
- 期：48
- 页码：15122-15134
- 全文大小：859K
- ISSN：1520-5126
文摘
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鈥?59) 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 F枚rster 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 伪 and that, upon binding, the 627 domain samples a dynamic conformational equilibrium in the vicinity of the C-terminus of importin 伪. 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.