Unraveling the roles of ammonium transporters and receptors through structural and functional characterization

Abstract: Ammonium transporters (Amts) play a pivotal role in nitrogen assimilation, facilitating the transport of ammonium (NH4+), the most reduced form of nitrogen, across cellular membranes. Amts are present across all kingdoms of life and have evolved to serve diverse functions, ranging from NH4+ transport to cellular homeostasis and detoxification. Amts exhibit a remarkable degree of conservation in critical residues essential for their function, as well as in their structural attributes, including a trimeric organization with each monomer featuring eleven or twelve membrane-spanning α-helices.
The anammox bacterium "Candidatus Kuenenia stuttgartiensis" has seven amt genes. Among them, five encode seemingly typical Amt proteins, while the remaining two, KsAmt4 and KsAmt5, possess outer-membrane domains. Inside the bacterium's specialized compartment, the anammoxosome, NH4+ is converted with nitrite, to dinitrogen. This process generates an electrochemical proton gradient used for ATP synthesis. Each of these amt genes is differentially expressed and have distinctive locations amongst its three unique and specific membranes.
In this work, unprecedented expression, isolation and purification of KsAmt1, KsAmt2 and KsAmt3 is attempted. Only KsAmt1 and KsAmt3 were successfully expressed. KsAmt3 was further purified and explored through electrophysiological and structural assays.
Employing solid supported membrane (SSM)-based electrophysiology experiments, we could show that KsAmt3 is transporting NH4+ across a membrane, displaying a selectivity that is characteristic of canonical Amts. Cryo-electron microscopy models unveil a stable Amt domain, juxtaposed with a highly flexible extended C-terminal region. While the Amt domain of this protein preserves all canonical features of Amt transporters, the extended C-terminal region is postulated to interact with the PII regulatory protein KsGlnK3, situated upstream of ksamt3. Although this work includes assays to detect complex formation between KsAmt3 and KsGlnK3, optimal conditions for this interaction remain elusive.
Notably, some Amts can function as ammonium receptors (AmRs), exemplified by KsAmt5, which encompasses a C-terminal histidine kinase (HK) transducer domain, along with an NH4+-binding site (N1/N2). This research investigates the impact of mutations at the N1/N2 site via SSM-based electrophysiology assays. Although the mutations induced slight decreases in NH4+ affinity, they did not impair NH4+ binding entirely, and transport was not regained. Additional activity assays elucidated the action mechanism of the HK domain, confirming its auto-kinase activity, dependent on NH4+ concentrations

Object Details

• Location: Deutsche Nationalbibliothek Frankfurt am Main
• Extent: Online-Ressource
• Language: Englisch
• Notes: Universität Freiburg, Dissertation, 2023

Classification and Topics

• Classification: Chemie
• Keyword: Carrier-Proteine

Contributors, Places and Time

• Event: Veröffentlichung
• (where): Freiburg
• (who): Universität
• (when): 2023
• Creator: Di Renzo López, Maria Agostina
• Contributor: Andrade, Susana ; Einsle, Oliver ; Köhn, Maja ; Albert-Ludwigs-Universität Freiburg. Fakultät für Chemie und Pharmazie

Further information

• DOI: 10.6094/UNIFR/242413
• URN: urn:nbn:de:bsz:25-freidok-2424131
• Rights: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Last update: 15.08.2025, 7:20 AM CEST