Modelling modulating phenomena for protein function in a serotonin-gated ion channel with molecular dynamics and enhanced sampling methods

Student thesis: Doctoral ThesisDoctor of Philosophy


Pentameric ligand-gated ion channels (pLGICs) are important proteins, embedded in the lipid mem-branes of nerve cells, which are of great pharmacological relevance. They mediate fast synaptic transmission and are involved in many diseases. For this reason, their fundamental understanding at an atomistic level is paramount for the design of new, targeted pharmaceuticals. Phenomena of interest include ligand binding/unbinding, opening and closing of the inner ion channel, mechanical signal propagation, allosteric modulation by lipids of the cell membrane and/or by other ligands, and more. Depending on the time scales of the specific phenomenon of interest, a variety of com-putational (or in-silico) techniques can be employed for their study. One of the most common is molecular dynamics, which involves the solution of Newton’s equations of motion for every particle that belongs to the system under investigation. This technique allows for the study of equilib-rium and out-of-equilibrium phenomena, typically in the range of nanoseconds-to-microseconds, depending on the size of the system (between orders of 103 atoms to orders of 106 atoms in the case of bio-systems). However, rare events are hardly sampled by the usage of molecular dynamics, thus requiring the introduction of the so called enhanced sampling techniques. Among these are metadynamics, umbrella sampling, steered molecular dynamics, and many others.
The goal of this PhD project is the investigation of several phenomena revolving around one pLGIC, namely the serotonin-activated type 3 A ion channel, or 5-HT3A receptor. The importance of this receptor is proven by its involvement in diseases such as schizophrenia, drug abuse, and many others. In this system, the binding of serotonin triggers a cascade of conformational changes that ultimately culminates with the opening of a hydrophobic gate, leading to a flow of ions through the inner ionic channel. This cascade of events typically occurs over a time window of milliseconds: in this context, molecular dynamics is an ideal tool to study single phenomena that make up the overall cascade. Its complete mechanism is, however, far from being fully understood. We focus here on studying phenomena that occur in different parts of this protein, and that, globally, contribute the overall mechanism of this molecular machine.
The first chapter of this thesis aims at providing all relevant information about pLGICs and relative ligands, with a particular focus on the 5-HT3A receptor, while the second chapter provides theoretical details on all-atom molecular dynamics and on the enhanced sampling methods used. The subsequent chapters describe the results obtained in each of the projects carried out during the PhD years, providing an introduction to the specific phenomena studied, their relevance, and a relative literature review.
Specifically, in the third chapter, we investigate the possibility that the propagation of mechan-ical signal across the extracellular and the transmembrane domains of the receptor is mediated by the isomerisation of a proline located at their interface. This consists in the rotation of a tor-sional angle linking this amino-acid and the subsequent residue. This study is carried out both with molecular dynamics and with metadynamics, with the aim of capturing structural differences that the different proline isomers induce in the surroundings, and of reconstructing the free en-ergy associated with the isomerisation process. In this way, we are able to assess how the protein environment and the proline isomerisation influence each other.
In the fourth chapter, we focus our attention to what happens at the interface between the protein transmembrane domain and the lipid membrane. In recent years, the modulation by the lipids (phospholipids and cholesterol in particular) onto the working mechanism of several pLGICs has been proved both in-silico and experimentally. In this context, our goal is to characterise, via molecular dynamics simulations, lipid-protein interactions, i.e. the binding of specific lipids to the surface of the protein, to assess the effects that lipids have on structural and dynamical properties of the protein, and to show how these are influenced by the exact concentrations of the lipid species.
In the fifth chapter, we investigate the role of the outermost M4 α helices of the transmembrane domain on the protein function. According to experimental results, disrupting crucial interactions in this section of the protein may alter the overall protein function. Here, we perform molecular dynamics simulations to assess how a specific mutation on the M4 helix, i.e. Y441A, is able to alter structural and dynamical properties of the M4 itself and of its surroundings, with the aim of shedding light on how the M4 is relevant for the mechanism of the serotonin-activated receptor.
In the sixth chapter, we present a preliminary study of the unbinding of serotonin from the orthosteric pocket, located in the extracellular domain of the receptor. We perform this study by means of both molecular dynamics and metadynamics simulations, with the aim of understanding the fundamental interactions involved in the process.
Overall, the original work carried out and explained in this thesis contributes to the fundamental understanding of the 5-HT3A receptor, and by extension of pLGICs in general.
Date of Award1 Jun 2021
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorCarla Molteni (Supervisor) & Chris Lorenz (Supervisor)

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