Abstract
Residence time, which refers to the average duration a drug remains bound to its receptor, is a crucial parameter in determining its pharmacological effects. However, the mechanisms governing the residence time of G protein-coupled receptor (GPCR) ligands remain unclear. In this study, we observed NMR signals from the methyl groups of alanine and methionine located at the intersection of the binding cavity and extracellular loops of A(2A)AR under conditions where E165Q and T256A mutations led to reduced residence times. Our NMR analysis revealed that the spatial arrangement surrounding the E165-H264 salt bridge correlates with residence time. These findings provide quantitative insights into residence time and could assist in the development of drugs with optimized effects.