Combining NMR and small angle X-ray and neutron scattering in the structural analysis of a ternary protein-RNA complex
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Many processes in the regulation of gene expression and signaling involve the formation of protein complexes involving multi-domain proteins. Individual domains that mediate protein-protein and protein-nucleic acid interactions are typically connected by flexible linkers, which contribute to conformational dynamics and enable the formation of complexes with distinct binding partners. Solution techniques are therefore required for structural analysis and to characterize potential conformational dynamics. Nuclear magnetic resonance spectroscopy (NMR) provides such information but often only sparse data are obtained with increasing molecular weight of the complexes. It is therefore beneficial to combine NMR data with additional structural restraints from complementary solution techniques. Small angle X-ray/neutron scattering (SAXS/SANS) data can be efficiently combined with NMR-derived information, either for validation or by providing additional restraints for structural analysis. Here, we show that the combination of SAXS and SANS data can help to refine structural models obtained from data-driven docking using HADDOCK based on sparse NMR data. The approach is demonstrated with the ternary protein-protein-RNA complex involving two RNA recognition motif (RRM) domains of Sex-lethal, the N-terminal cold shock domain of Upstream-to-N-Ras, and msl-2 mRNA. Based on chemical shift perturbations we have mapped protein-protein and protein-RNA interfaces and complemented this NMR-derived information with SAXS data, as well as SANS measurements on subunit-selectively deuterated samples of the ternary complex. Our results show that, while the use of SAXS data is beneficial, the additional combination with contrast variation in SANS data resolves remaining ambiguities and improves the docking based on chemical shift perturbations of the ternary protein-RNA complex.
KeywordsNMR SANS SAXS Protein-RNA complexes Integrated structural biology
We are grateful to the ILL for BAG SANS beamtime on D22 (local contact Dr. Anne Martel) and the ESRF for beamtime on BM29 (local contact Dr. Louiza Zerrad). We thank Dr. Fatima Gebauer (CRG Barcelona, Spain) for plasmids and helpful discussions. Janosch Hennig acknowledges postdoctoral fellowships by the Swedish Research Council (VR, Vetenskapsrådet) and the European Molecular Biology Organization (EMBO, ALTF 276-2010). This work was supported by the Deutsche Forschungsgemeinschaft (SFB1035, GRK1721 to M.S.) and the European Commission (FP7 NMI3 project, to M.S.). The authors declare that they have no conflict of interest.
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