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.

Combining molecular dynamics simulations with small-angle X-ray and neutron scattering data to study multi-domain proteins in solution

“…As mentioned in the introduction, there are several ways to combine MD simulations with SAS data (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), but we here used the Bayesian/Maximum Entropy (BME) method (3) and the above-calculated SAXS and SANS intensities to reweight the trajectories. For details of BME see (3) as well as code and examples online https://github.com/KULL-Centre/BME.…”

“…In that case, however, simulations and experiments may be used synergistically to generate and refine the description of flexible molecules. Thus, as described by us and others, SAXS and molecular simulations can be combined to determine a structural ensemble that represents the system, and is compatible with the information in the force field and the experimental constraints from SAXS (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20).…”

Single Pair Förster Resonance Energy Transfer: A Versatile Tool To Investigate Protein Conformational Dynamics

“…Several FRET pairs typically used for two‐color FRET experiments have evolved. These include Cy3/Cy5, Atto532/Atto647N, Atto532/Atto647, Atto532/Alexa647, Alexa488/Alexa594, Alexa488/Cy5, and Alexa488/Alexa647 . For an overview of fluorophores used in spFRET experiments, see Box .…”

Multiple frequency saturation pulses reduce CEST acquisition time for quantifying conformational exchange in biomolecules

“…The former method can be employed when the populations between states are comparable, while the latter methods are useful when a minor population is in exchange with the major conformational state 14 or where the molecule is in exchange with a large molecular weight species that is unobservable by solution NMR 16 . Biological processes occurring on this timescale include conformational changes needed for molecular transport 17–19 , protein folding 20–22 , enzyme catalysis 23 , and molecular recognition mechanisms 24,25 .…”