Fast Quantitative Validation of 3D Models of Low-Affinity Protein–Ligand Complexes by STD NMR Spectroscopy

Low-affinity protein–ligand interactions are important for many biological processes, including cell communication, signal transduction, and immune responses. Structural characterization of these complexes is also critical for the development of new drugs through fragment-based drug discovery (FBDD), but it is challenging due to the low affinity of fragments for the binding site. Saturation transfer difference (STD) NMR spectroscopy has revolutionized the study of low-affinity receptor–ligand interactions enabling binding detection and structural characterization. Comparison of relaxation and exchange matrix calculations with 1H STD NMR experimental data is essential for the validation of 3D structures of protein–ligand complexes. In this work, we present a new approach based on the calculation of a reduced relaxation matrix, in combination with funnel metadynamics MD simulations, that allows a very fast generation of experimentally STD-NMR-validated 3D structures of low-affinity protein–ligand complexes.