Abstract
Ion channels are fundamental units of the nervous system responsible for mediating intercellular signals and propagating action potentials, this makes them critical in brain function, disease, and pharmacology. Pentameric Ligand Gated Ion Channels (pLGICs) are a subgroup of the proteins present at synapses responsible for modulating synaptic activity; the glycine receptor (GlyR) is a common variant responsible for inhibitory action. Understanding the activation of these channels requires detailed structural and functional profiling, which is limited by current experimental techniques. Structural data can be collected to a very high resolution of under 3 Å, and functional data can be generated with electrophysiology experiments to determine channel opening times and binding rates. However, the structure can only be observed in snapshots of time after being modified and processed in non-physiological conditions, where functional data and dynamics cannot be simultaneously collected. Atomistic simulations can overcome these limitations by providing atomic resolution dynamics on timescales of hundreds of ns to ms. In this PhD project, we investigated the binding of the glycine receptor through the application of these techniques on various systems.Three systems of the GlyR-α1 were developed to provide insights into the binding pocket dynamics of pLGICs, one bound with glycine to represent the endogenous ligand binding, a system with the partial agonist GABA bound and a system with the N46K mutation. Through simulating these systems with molecular dynamics and metadynamics, we can carry out comparative analysis across them, focusing on the binding profiles and binding/unbinding events. The aim of this PhD is to utilise this analysis to gain key insights into the binding dynamics of GlyRs with a focus on the binding mode, the differences that affect binding affinity so potently between partial and full agonists, and how mutants can impact the binding mode of pLGICs. The first chapter of this thesis will cover the essential context and required for understanding both the function and structure of pLGICs and specifically the GlyRs, as well as an introduction to partial versus full agonism, mutants of interest in GlyRs and the techniques used to study these channels experimentally and computationally. The second chapter will describe and detail the theoretical concepts and tools used for the work in later chapters covering all atom molecular dynamics, metadynamics and funnel metadynamics.
The third chapter covers the development of the GlyR system and an investigation of the binding mode in unbiased simulations. This chapter includes a comparison of a whole protein membrane embedded system against an extracellular domain system along with an analysis of the starting conditions, quality, and stability of the binding during simulations. The fourth chapter provides a further analysis of the binding mode in the context of full and partial agonism, expanding upon unbiased simulations through the application of funnel metadynamics to profile and compare the free energy surface and binding paths of full and partial agonists. The fifth chapter applies the same approach and methods but for comparing a wild type system versus the N46K mutant variant to establish an atomistic description of how this mutation impacts ligand binding. Overall, the work displayed and discussed in this thesis provides an expanded view into the dynamics of GlyR binding and consequently a clearer understanding of pLGICs.
| Date of Award | 1 Jul 2024 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Carla Molteni (Supervisor) & Lucia Sivilotti (Supervisor) |