Small-molecule binding sites on proteins established by paramagnetic NMR spectroscopy

Area of Science:

• Biophysics
• Structural Biology
• Chemical Biology

Background:

• Determining the 3D structure of small molecule-protein complexes with weak interactions is challenging.
• Weak affinity complexes are crucial in drug discovery, particularly in early-stage fragment-based approaches.
• Existing structural biology methods often struggle with low-affinity interactions.

Purpose of the Study:

• To present a novel paramagnetic Nuclear Magnetic Resonance (NMR) method for structural analysis of weak-affinity protein-ligand complexes.
• To enable the determination of intermolecular structure restraints using pseudocontact shifts (PCSs).
• To facilitate fragment-based drug discovery by providing structural information on initial fragment hits.

Main Methods:

• Utilized paramagnetic tags attached to the protein surface to generate pseudocontact shifts (PCSs) in the ligand.
• Measured ligand PCSs from simple 1D proton (1H) spectra.
• Employed PCSs as docking restraints for structure determination.
• Validated structures using intermolecular Nuclear Overhauser Effects (NOEs).

Main Results:

• The paramagnetic NMR method successfully determined structural restraints for weak-affinity complexes.
• Structures derived from PCSs and NOEs showed good agreement.
• Accurate prediction of magnetic susceptibility tensors with two-armed probes allowed low-resolution modeling without isotope-labeled protein.
• The method is suitable for ligands with dissociation constants in the high micromolar to millimolar range.

Conclusions:

• Paramagnetic NMR-based PCSs provide a robust method for structural elucidation of weak-affinity protein-ligand complexes.
• This approach is particularly valuable for fragment-based drug discovery, offering structural insights early in the screening process.
• The method's ability to function with low ligand-bound fractions and without isotope-labeled proteins enhances its applicability.