Ion-water coupling controls class A GPCR signal transduction pathways

Abstract

G-protein-coupled receptors (GPCRs) are membrane proteins that transmit signals across the cell membrane by activating intracellular G-proteins in response to extracellular ligand binding. A large majority of GPCRs are characterised by an evolutionarily conserved activation mechanism, involving the re-orientation of helices and the conformation of key residue side chains, rearrangement of an internal hydrogen bonding network, and the expulsion of a sodium ion from a binding site in the transmembrane region. However, how sodium, internal water, and protein residues interplay to determine the overall receptor state remains elusive. Here, we develop and apply “State Specific Information” (SSI), a novel methodology based on information theory, to resolve signal transmission pathways through the proteins. Using all-atom molecular dynamics simulations of pharmaceutically important GPCRs, we find that sodium plays a causal role in the formation and regulation of a communication channel from the ion binding site to the G-protein binding region. Our analysis reveals that the reorientation of specific water molecules is essential to enable coupled conformational state changes of protein residues along this pathway, ultimately modulating the G-protein binding site. Furthermore, we show that protonation of the ion binding site creates a conformational coupling between two previously separate motifs, entirely controlled by the orientation of two water molecules, priming the receptor for activation. Taken together, our results demonstrate that sodium serves as a master switch, acting in conjunction with the network of internal water molecules, to determine the micro- and macrostates of GPCRs during the receptors’ transition to activation.

A co-operative knock-on mechanism underpins Ca2+-selective cation permeation in TRPV channels

“…In order to go beyond just visual inspection of the trajectories and to assess the co-operativity of ion permeation in a quantitative way, we developed a new approach based on mutual information, taking into account the “state” of each ion binding site. To achieve this, we assigned a specific binding state (unoccupied, or occupied with a specific ion) to binding sites A, B, and C and used our recently developed approach, state-specific information (SSI, (52)) on pairs of adjacent, permeating cations, to quantify the degree of coupling between ion binding transitions at each of these sites (see Methods ). This analysis yields a coefficient quantifying the co-operativity between ion binding and unbinding at neighbouring or more distant binding sites, where a greater coefficient signifies a higher degree of coupling; which suggests that when an ion transitions from one site it is more likely there is a transition at the other.…”

“…We hypothesised that the level of co-operativity between sites A, B and C in the pore could underpin the difference between highly and less Ca 2+ -selective TRPV channels. We therefore developed a novel method to quantify co-operativity during ion permeation in pores with multiple ion binding sites based on mutual information and total correlation measures using the SSI approach (52). We anticipate that this method will be similarly useful for the study of permeation mechanisms and the basis of selectivity in other channels.…”

“…Characterising permeation co-operativity through mutual information using SSI from PENSA. To characterise the level of co-operativity in the knock-on permeation mechanisms in TRPV channels, we used PENSA to calculate the state-specific information (SSI) shared between discrete state transitions in the occupancy distributions of each binding site (44,52).…”

Hydration Dynamics in Biological Membranes: Emerging Applications of Terahertz Spectroscopy

“…36 These studies, along with reports correlating the formation of a continuous internal water pathway 37 and water influx into the receptor cavity 38 with GPCR activation, has led to the recognition of membrane hydration dynamics as an important parameter in the mechanistic framework of GPCR-mediated signal transduction. 39 Interestingly, a recent cryo-EM structure of the clinically relevant serotonin 1A neurotransmitter GPCR 40−43 has shown the presence of "structured water molecules" in the ligand-binding pocket that resemble water around the natural ligand (serotonin) of this receptor subtype, 44 which could contribute to the long-standing discourse on the origin of basal activity (i.e., activity in the absence of ligands) in GPCRs. Importantly, the reduced therapeutic response of a clinical GPCR variant implicated in respiratory diseases has been ascribed to the loss of a water-mediated network crucial for stabilizing the ligand binding site, 45 thereby hinting at the potential application of hydration signatures as molecular markers in pathophysiological conditions (see below).…”

“…In addition, GPCR activation has been demonstrated to trigger extension of such a protein–water hydrogen-bond network, bridging multiple sequence/structural motifs crucial for signal transduction, throughout the receptor lumen . These studies, along with reports correlating the formation of a continuous internal water pathway and water influx into the receptor cavity with GPCR activation, has led to the recognition of membrane hydration dynamics as an important parameter in the mechanistic framework of GPCR-mediated signal transduction . Interestingly, a recent cryo-EM structure of the clinically relevant serotonin 1A neurotransmitter GPCR − has shown the presence of “structured water molecules” in the ligand-binding pocket that resemble water around the natural ligand (serotonin) of this receptor subtype, which could contribute to the long-standing discourse on the origin of basal activity (i.e., activity in the absence of ligands) in GPCRs.…”