Hydration Drives Activation of the G‐Protein‐Coupled Receptor Rhodopsin

Abstract

G-protein-coupled receptors (GPCRs) are a pivotal superfamily of seven-helix transmembrane receptors responsible for the transduction of stimuli across cellular membranes. They are the targets of ~30% of the pharmaceuticals currently in the market. Understanding how the soft membrane matter (lipids and water) influences the structural changes during the activation of GPCRs is crucial to discovering and developing novel GPCR-targeted drugs [1]. Here, we assessed the effects of hydration on the conformational dynamics of GPCRs by subjecting the archetypical GPCR-rhodopsin in native retinal disk membranes to varying osmotic pressures using polyethylene glycol (PEG) solutions of different molar mass [2,3]. The metastable activation equilibrium of rhodopsin (Keq) after photoexcitation was quantified using UV-visible spectroscopy. Analysis of the results using thermodynamic relations revealed for the first time an influx of ~80-100 water molecules flooding into the rhodopsin interior during photoactivation, validating previous molecular dynamics (MD) simulations [4]. Both applied osmotic pressure and the osmolyte size affected the metarhodopsin equilibrium. Dehydrating conditions generally favored the closed inactive Meta-I state through an efflux of water from the protein interior, while small penetrating osmolytes stabilized the expanded active Meta-II conformation in low concentration regimes. The pH titration curves for the metarhodopsin equilibrium in the presence of various PEG osmolytes showed that hydration is coupled to activation via modulation of the pKA of Glu134 of the conserved E(D)RY sequence motif. By changing water availability, osmotic stress perturbs the dielectric constant characterizing the Glu134 microenvironment and the thermodynamics of its protonation. Activation of rhodopsin is contingent upon the change in electrostatic microenvironments by rearranging water molecules within the protein conformation, dramatically recasting the role of soft matter in biological signal transduction. [1] M.F. Brown (2017) Annu. Rev. Biophys. 46, 379-410. [2] U. Chawla et al. (2020) Angew. Chem. Int. Ed. doi:10.1002/anie.202003342. [3] N. Weerasinghe et al. (2018) Biophys. J. 114, 274a. [4] N. Leioatts et al. (2014) Biochemistry 53, 376−385.