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

Area of Science:

• Structural biology
• Biophysics
• Drug discovery

Background:

• Protein-ligand interactions involve conformational changes measurable by solution small-angle X-ray scattering (SAXS).
• SAXS titrations can quantify interaction strength but are underutilized in drug discovery due to uncertainty in sample requirements and prediction accuracy.

Purpose of the Study:

• To benchmark ligand screening performance using SAXS titrations.
• To establish reliable protocols for SAXS-based ligand screening in drug discovery.

Main Methods:

• Utilized histidine-binding protein and other periplasmic binding proteins for benchmarking.
• Varied sample concentrations and exposure times across multiple synchrotron beamlines.
• Analyzed volatility ratios between titrated and apo reference scattering curves.

Main Results:

• The volatility ratio effectively captures structural and population changes, simplifying analysis.
• Achieved affinity prediction accuracy comparable to isothermal titration calorimetry under specific conditions (Kd within 10^2 of protein concentration, >20s total exposure).
• Optimized protocol at 0.5 mg/mL concentration.

Conclusions:

• SAXS titrations provide a robust and accurate method for quantifying protein-ligand interactions.
• The developed protocol enables high-throughput screening with an estimated 20-100 ligand titrations per day.
• This approach enhances the utility of SAXS in drug discovery workflows.