G-protein–coupled receptors (GPCRs) are the largest family of membrane proteins in the human genome and act as signal transducers that enable transmembrane communication between cells and membrane-bound organelles. Despite many GPCR structures having water molecules within their transmembrane regions, the role that solvent molecules play in the receptor dynamics and signaling behavior remains unexplored. Here we show experimentally that activation of the archetypical GPCR rhodopsin in the lipid membrane is slaved to bulk water movements into the protein (Angew.Chem.Int.Ed. 2021, 60, 2288). To quantify the changes in hydration of the receptor during activation, we measured reversible shifting of the metarhodopsin equilibrium due to osmotic stress using polyethylene glycol osmolytes. Besides generating osmotic pressure due to their concentrations, the size of the osmolytes also influenced the rhodopsin activation. We discovered that light activation entails a large influx of bulk water (80–100 molecules) into the receptor, giving new insight into the GPCR activation mechanism. Large solutes are excluded from rhodopsin and dehydrate the protein, favoring the inactive Meta-I state. By contrast, small osmolytes initially shift the equilibrium toward the active Meta-II state until a quantifiable saturation point is reached, similar to gain-of-function protein mutations. In the limit of increasing osmolyte size, a universal response of rhodopsin to osmotic stress is observed, suggesting the protein adopts a dynamic, hydrated sponge-like state upon photoactivation. Our results demand a rethinking of the role of solvent water dynamics in modulating various intermediates in the GPCR energy landscape. Besides structural and bound water, we propose that an influx of bulk water plays a necessary role in establishing the active GPCR conformation that mediates signaling. Our results dramatically recast the role of cellular water from spectator molecule to direct allosteric regulator in GPCR signaling.

G-protein–coupled receptor activation by hydration-driven sponge mechanism

ACS Spring 2022

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