Structural, functional and mechanistic analysis of the Escherichia coli ammonium transporter AmtB

The exchange of ammonium across cellular membranes is an essential process in all kingdoms of life. Ammonium is a major source of nitrogen for bacteria, fungi, and plants, whereas in animal cells it is a cytotoxic waste metabolite, which must be excreted. The transport of ammonium is accomplished by the ubiquitous Ammonium transporter/Methylammonium permease/Rhesus (Amt/Mep/Rh) superfamily of membrane proteins. Inaddition to their fundamental role in facilitating the transport of metabolites, members of the Amt/Mep/Rh superfamily are important drivers of the virulence of infectious fungi. In humans, malfunctions of Rh proteins are linked toinherited haemolytic anaemia, stomatocytosis, and early onset depressive disorder, amongst many other human pathologies. Therefore, an improved understanding of the function of ammonium transporters has importantmedical implications alongside the elucidation of a central biological process. In spite of its general importance, a consensus on the pathway and mechanism of ammonium transport by this superfamily has not yet been achieved.The overall aim of this project is to use Escherichia coli AmtB, the paradigmatic, most intensely studied member of the Amt/Mep/Rh protein family and an integrative approach combining molecular genetics, biochemistry, biophysics and molecular dynamic simulations to gain valuable structural and functional information on this important protein family. A new in-solution structural approach combining Small Angle Neutron/X-ray Scattering with Molecular Dynamics simulation has been successfully developed providing a new tool to study membrane protein dynamics insolution. Moreover, a new mechanism for the deprotonation/translocation associated with ammonium transport through AmtB was suggested and demonstrated by using Solid Supported Membrane Electrophysiology (SSME)and Molecular Dynamics simulations on genetic variants. Furthermore, the influence of 1-palmitoyl-2-oleoyl phosphatidylglycerol (PG), a specific lipid known to structurally interact with AmtB, was assessed and it was suggested that this lipid is essential for translocation of ammonium across AmtB. Last but not least, the electrophysiological technique developed on AmtB has been extended to study the bacterial Rh and fungal Mep proteins to explore whetherthe mechanism I propose for AmtB is a common feature for the Amt/Mep/Rh protein family. Given the importance of this protein family in various fundamental biological processes and human diseases, this work may, in the long term, lead to the development of new therapeutic interventions.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral-pg

Grantor dc:publisher.institution

University of Strathclyde

Year dc:date.issued

2018

Author dc:creator

• Mirandela, Gaëtan Dias

Advisors dc:contributor.advisor

• Hoskisson, Paul A.
• Javelle, Arnaud

DOI

10.48730/h278-mp73

Identifier

T15111

Author Identifier

201583137

OAI identifier oai:identifier

oai:strathclyde:1z40ks86n