Oral Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Using small-angle X-ray scattering to screen for and classify biomolecular interactions (#6)

Po-chia Chen 1 , Pawel Masiewicz 1 , Vladimir Rybin 1 , Dmitri Svergun 2 , Janosch Hennig 1
  1. European Molecular Biology Laboratory, Heidelberg, Heidelberg, BADEN-WURTTEMBERG, Germany
  2. EMBL Hamburg, Hamburg, Germany

The structural characterisation of many RNA-binding proteins (RBPs) requires us to address two particular challenges: One, the presence of both sequence-specific and non-specific interactions due to the ubiquitous presence of charged contacts. Two, given the modular nature of many RBPs as a series of small domains, discerning the role of each domain in the overall function. These two issues increase the likelihood of multiple binding modes in any given interaction, which complicate screening efforts to identify strong-affinity RNA sequences (also known as cognate motifs,) required to apply structural biology techniques. While existing affinity-based screens provide strong leads for candidate motifs, the cost of production of specific RNA sequences renders their validation significantly less feasible using techniques techniques such as ITC and NMR.

We report here a SAXS-based screening workflow (SAXScreen) that estimates binding affinity and categorises binding mechanisms based on measured intensities over ligand titrations alone, in order to preserve basic applicability to other biomolecular interactions. Using synchrotron sources to provide cost-effective material consumption, SAXScreen is validated using the two RRM domain Sex-lethal protein and a sparse search around its cognate poly-uridine motif. Titration over ~35 RNA motifs reveal sequence-dependent variations in complex formation, which in combination with structural modelling is shown to be based on preferences between 1:1 and 2:2 binding stoichiometries. Further confirmation via NMR titration differentiates between the roles of domains RRM1 and RRM2, as well as specific interactions with poly-uridines versus non-specific interactions with poly-cytidines. These findings position SAXScreen as a complementary screening tool that provides preliminary structural characterisation of biomolecular interactions.