Mechanistic characterisation of the Escherichia coli ammonium transporter AmtB

  • Gordon David Williamson

Student thesis: Doctoral Thesis

Abstract

The exchange of ammonium across cellular membranes is a fundamental process in all domains of life. In plants, bacteria, and fungi, ammonium represents a vital nitrogen source, which they seek to scavenge from the external environment. In contrast, ammonium is a cytotoxic metabolic waste product in animal cells and must be excreted to prevent cell death. Transport of ammonium is facilitated by the ubiquitous Amt/Mep/Rh transporter superfamily. In addition to their function as transporters, Amt/Mep/Rh proteins play roles in a diverse array of biological processes. For example, Mep proteins signal the onset of pseudohyphal growth, a transition associated with virulence in pathogenic fungi. The human Rh proteins are also essential in maintaining acid-base homeostasis, and their malfunction can lead to various pathologies, including hereditary anaemias, overhydrated stomatocytosis, and early-onset depressive disorders. Despite this clear physiological importance, the mechanism of Amt/Mep/Rh proteins has remained elusive. Crystal structures of AmtB from Escherichia coli, the most intensely studied member of the family, suggest electroneutral transport, whilst functional evidence supports an electrogenic mechanism. The overall goal of this project was to combine electrophysiology, yeast functional complementation, and extended molecular dynamics simulations (MDS) to characterise the mechanism of ammonium transport in AmtB.;An in vitro assay based on Solid Supported Membrane Electrophysiology (SSME) was developed to confirm electrogenic activity in AmtB and characterise activity, selectivity, and kinetics of WT AmtB (Chapter 3). MDS revealed two ordered water chains embedded within the pore of AmtB, representing a potential polar transfer network. Subsequent SSME and in vivo yeast complementation characterisation of AmtB variants demonstrated that these wires were vital for AmtB-mediated NH4+ transport. This led to the proposal of a novel mechanism wherein NH4+ is deprotonated and H+ and NH3 are carried separately across the membrane (Chapter 4).;Disruption of the twin-His motif, a highly conserved histidine dyad within the pore of AmtB, had a significant impact on the kinetics of ammonium transport and a deleterious impact on selectivity, resulting in passage of potassium ions through AmtB. It is imperative that transporters maintain substrate selectivity, as uncontrolled entry of charged molecules can prove fatal to the cell, thus this explains the conservation of the twin-His motif within the Amt/Mep/Rh family (Chapter 5).;This work provides a novel model of AmtB-mediated electrogenic transport and offers valuable insight into the mechanism of the highly important Amt/Mep/Rh family. Given their physiological relevance, this work may form the foundation for future medical interventions and treatments.
Date of Award1 Jan 2021
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SponsorsUniversity of Strathclyde & EPSRC (Engineering and Physical Sciences Research Council)
SupervisorArnaud Javelle (Supervisor) & Iain Hunter (Supervisor)

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