Structure-based screening of binding affinities via small-angle X-ray scattering

ABSTRACT Protein-protein and protein-ligand interactions can alter the scattering properties of participating molecules, and thus be quantified by solution small-angle X-ray scattering (SAXS). In such cases, scattering reveals structural details of the bound complex, number of species involved, and in principle strength of the interaction. However, determining binding affinities from SAXS-based titrations is not yet an established procedure with well-defined performance expectations. We thus used periplasmic binding proteins and in particular histidine-binding protein as a standard reference, then examined precision and accuracy of affinity prediction at multiple concentrations and exposure times. By analyzing several structural and comparative scattering metrics, we found that the volatility of ratio between titrated scattering curves and a common reference most reliably quantifies ligand-triggered changes. This ratio permits the determination of affinities at low signal-to-noise ratios and without pre-determining the complex scattering, demonstrating that SAXS-based ligand screening is a promising alternative biophysical method for drug discovery pipelines. SIGNIFICANCE Solution X-ray scattering can be used to screen a set of biomolecular interactions, which yields quantitative information on both structural changes and dissociation constants between binding partners. However, no common benchmarks yet exist for the application of SAXS within drug discovery workflows. Thus, investigations into its performance limitations are currently needed to make SAXS a reliable source for high-throughput screening. This study establishes a generalizable protocol based on protein-ligand interactions, and demonstrates its reproducibility across several beamline setups. In the simplest case, the micromolar binding affinities can be determined directly from measured intensities without knowledge of the molecular structure, with material consumption that is competitive with other biophysical screening techniques.