tetano
Editor, Senior Moderator
J Am Chem Soc. 2015 Oct 1. [Epub ahead of print]
[h=1]Large Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α.[/h] Delaforge E, Milles S, Bouvignies G, Bouvier D, Boivin S, Salvi N, Maurin D, Martel A, Round A, Lemke EA, Jensen MR, Hart D, Blackledge M.
[h=3]Abstract[/h] 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 and x-ray scattering and 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 (CEST) 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 [PubMed - as supplied by publisher]
[h=1]Large Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α.[/h] Delaforge E, Milles S, Bouvignies G, Bouvier D, Boivin S, Salvi N, Maurin D, Martel A, Round A, Lemke EA, Jensen MR, Hart D, Blackledge M.
[h=3]Abstract[/h] 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 and x-ray scattering and 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 (CEST) 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 [PubMed - as supplied by publisher]