Lysine 101 in the CRAC Motif in Transmembrane Helix 2 Confers Cholesterol-Induced Thermal Stability to the Serotonin Receptor.

G protein-coupled receptors (GPCRs) constitute the largest class of membrane proteins that transduce signals across the plasma membrane and orchestrate a multitude of physiological processes within cells. The serotonin receptor is a crucial neurotransmitter receptor in the GPCR family involved in a multitude of neurological, behavioral and cognitive functions. We have previously shown, using a combination of experimental and simulation approaches, that membrane cholesterol acts as a key regulator of organization, dynamics, signaling and endocytosis of the serotonin receptor. In addition, we showed that membrane cholesterol stabilizes the serotonin receptor against thermal deactivation. In the present work, we explored the molecular basis of cholesterol-induced thermal stability of the serotonin receptor. For this, we explored the possible role of the K101 residue in a cholesterol recognition/interaction amino acid consensus (CRAC) motif in transmembrane helix 2 in conferring the thermal stability of the serotonin receptor. Our results show that a mutation in the K101 residue leads to loss in thermal stability of the serotonin receptor imparted by cholesterol, independent of membrane cholesterol content. We envision that our results could have potential implications in structural biological advancements of GPCRs and design of thermally stabilized receptors for drug development.