Side-chain dynamics of the α1B-adrenergic receptor determined by NMR via methyl relaxation

G protein-coupled receptors (GPCRs) are medically important membrane proteins that sample inactive, intermediate, and active conformational states characterized by relatively slow interconversions (similar to mu s-ms). On a faster timescale (similar to ps-ns), the conformational landscape of GPCRs is governed by the rapid dynamics of amino acid side chains. Such dynamics are essential for protein functions such as ligand recognition and allostery. Unfortunately, technical challenges have almost entirely precluded the study of side-chain dynamics for GPCRs. Here, we investigate the rapid side-chain dynamics of a thermostabilized alpha(1B)-adrenergic receptor (alpha(1B)-AR) as probed by methyl relaxation. We determined order parameters for Ile, Leu, and Val methyl groups in the presence of inverse agonists that bind orthosterically (prazosin, tamsulosin) or allosterically (conopeptide rho-TIA). Despite the differences in the ligands, the receptor's overall side-chain dynamics are very similar, including those of the apo form. However,rho-TIA increases the flexibility of Ile176(4x56) and possibly of Ile214(5x49), adjacent to Pro215(5x50) of the highly conserved (PIF6x44)-I-5x50-F-3x40 motif crucial for receptor activation, suggesting differences in the mechanisms for orthosteric and allosteric receptor inactivation. Overall, increased Ile sidechain rigidity was found for residues closer to the center of the membrane bilayer, correlating with denser packing and lower protein surface exposure. In contrast to two microbial membrane proteins, in alpha(1B)-AR Leu exhibited higher flexibility than Ile side chains on average, correlating with the presence of Leu in less densely packed areas and with higher protein-surface exposure than Ile. Our findings demonstrate the feasibility of studying receptor-wide side-chain dynamics in GPCRs to gain functional insights.

Automated summary

This study investigates the rapid side-chain dynamics of a thermostabilized alpha(1B)-adrenergic receptor (alpha(1B)-AR) using methyl relaxation. The results show that the overall side-chain dynamics of the receptor are similar regardless of the ligands, but the allosteric ligand increases the flexibility of specific residues, suggesting differences in the inactivation mechanisms of orthosteric and allosteric receptors. Additionally, residues closer to the center of the membrane bilayer exhibit increased side-chain rigidity.