Exploring an Intracellular Allosteric Site of CC-Chemokine Receptor 4 from 3D Models, Probe Simulations, and Mutagenesis

We applied our previously developed probe confined dynamic mapping protocol, which combines enhanced sampling molecular dynamics (MD) simulations and fragment-based approaches, to identify the binding site of GSK2239633A (N-[[3-[[3-[(5-chlorothiophen-2-yl)sulfonylamino]-4-methoxyindazol-1-yl]methyl]phenyl]methyl]-2-hydroxy-2-methylpropanamide), a selective CC-chemokine receptor type 4 (CCR4) negative allosteric modulator, using CCR4 homology and AlphaFold models. By comparing the performance across five computational models, we identified conserved (K310^8.49 and Y304^7.53) and non-conserved (M243^6.36) residue hotspots for GSK2239633A binding, which were validated by mutagenesis and bioluminescence resonance energy transfer assay. Further analysis of 3D models and MD simulations highlighted the pair of residues 6.36 and 7.56 that might account for antagonist selectivity among chemokine receptors. Our in silico protocol provides a promising approach for characterizing ligand binding sites in membrane proteins, considering receptor dynamics and adaptability and guiding protein template selection for ligand design.