The interplay of ion binding, receptor ionisation, and water molecules in the regulation of G-protein coupled receptor signalling

: The development and application of a methodology to characterise protein-internal water and ion cavities and their mechanistic relationships from a mutual information perspective

Abstract

Owing to their crucial role in a vast array of biological processes, G-protein coupled receptors (GPCRs) form the primary drug target for over a third of all FDA approved drugs. While these transmembrane receptors have been the subject of immense scientific interest over the past few decades, much remains to be understood about the mechanistic underpinnings of how they perform agonist-induced transmembrane signal transduction. What is known is that environmental factors including; ion concentrations, transmembrane voltage, pH, and hydration levels, are able to allosterically modulate the receptors signalling states. A wealth of possible therapeutic avenues can be further explored by tapping into the fine details of how these environmental factors affect the GPCR activation mechanism. However, how these elements might intertwine to accomplish allosteric modulation of the receptors signalling state remains unknown.

Here, teamed with the development and application of a new methodology to characterise protein internal water molecules and analyse their relationships to ion-binding events and receptor dynamics, I present atomistic-detail computational simulations that suggest a mechanism of allosteric modulation, tying together ions, waters, and ligands with receptor dynamics. These simulations, which focus on the most abundant GPCR subfamily, the class A receptors, demonstrate that the function of ions and waters is intimately weaved together and conserved in multiple receptor types. I demonstrate that individual water molecules act as molecular switches, coupled to and activated by ion-binding events, which tune the receptors internal water-mediated polar network. Ion-binding events further govern the coordination of water pathways that connect multiple functionally relevant ionisable residues.

Class A GPCRs are characterised by the coordination of a Na⁺ ion at a highly conserved and ionisable binding site in the transmembrane domain. The presence of the ion is known to reduce the levels of agonist affinity and the levels of basal signalling, thus stabilising the receptor in an inactive signalling state. I reveal that Na⁺ inhibits a water pathway between two ionisable residues where counterbalanced protonation events are thought to be characteristic of high affinity agonist binding events and receptor activation. I hypothesise that this pathway could allow concerted loss and gain of a proton between these two residues, explaining how Na⁺ decreases the binding affinity of the agonist. The mechanistic underpinning of this inhibition is the ion-water coupled induction of a hydrophobic packing between conserved motifs, which stretches down the transmembrane domain and stabilises the signal protein binding cavity in a closed, inactive receptor state. I hypothesise that this Na⁺-induced hydrophobic packing diminishes basal signalling by restricting the dynamics of the signalling site.

Global rearrangement of transmembrane helices, such that they open up a G-protein binding cavity, is an essential step in receptor activation. Behind the large-scale dynamics are complex changes in an internal water-mediated polar network that couple to the formation of a transmembrane water channel. In conjunction with this is an intracellular exit of the Na⁺ ion, while protonation of the primary Na⁺ coordinating residue is understood to move the receptor toward the active signalling state. I report a synergistic relationship between two distal protonatable aspartates, which couple protonation events to the fine tuning of a single spatially conserved water molecule. The water molecule functions as a proton-coupled molecular switch that locks and unlocks a highly conserved hydrophobic residue, which is well known to gate the transmembrane water channel. In the μ-opioid receptor, protonation of both aspartates destabilises the hydrophobic gate, so that the dynamics of receptor motifs, internal hydration levels, and interhelical distances begin to sample the active signalling state. This thesis demonstrates that GPCRs harness a complex and highly adaptive ion-water coupling to move between activation states.

Date of Award	2023
Original language	English
Awarding Institution	University of Dundee
Sponsors	Biotechnology and Biological Sciences Research Council
Supervisor	Ulrich Zachariae (Supervisor) & Andrei Pisliakov (Supervisor)