Journal of Biomolecular NMR

, Volume 56, Issue 1, pp 17–30 | Cite as

Combining NMR and small angle X-ray and neutron scattering in the structural analysis of a ternary protein-RNA complex

  • Janosch Hennig
  • Iren Wang
  • Miriam Sonntag
  • Frank Gabel
  • Michael Sattler
Article

Abstract

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.

Keywords

NMR SANS SAXS Protein-RNA complexes Integrated structural biology 

Supplementary material

10858_2013_9719_MOESM1_ESM.pdf (5.9 mb)
Supplementary material 1 (PDF 6028 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Janosch Hennig
    • 1
    • 2
  • Iren Wang
    • 1
    • 2
  • Miriam Sonntag
    • 1
    • 2
  • Frank Gabel
    • 3
  • Michael Sattler
    • 1
    • 2
  1. 1.Institute of Structural Biology, Helmholtz Zentrum MünchenNeuherbergGermany
  2. 2.Center for Integrated Protein Science Munich at Chair Biomolecular NMR Spectroscopy, Department ChemieTechnische Universität MünchenGarchingGermany
  3. 3.Extremophiles and Large Molecular Assemblies Group (ELMA), Institut de Biologie Structurale (IBS) CEA-CNRS-UJFGrenobleFrance

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