The lipid environment determines the activity of theE. coliammonium transporter, AmtB

The movement of ammonium across biological membranes is a fundamental process in all living organisms and is mediated by the ubiquitous Amt/Mep/Rh family of transporters. Recent structural analysis and coupled mass spectrometry studies have shown that the Escherichia coli ammonium transporter, AmtB, specifically binds phosphatidylglycerol (PG). Upon PG binding, several residues of AmtB undergo a small conformational change, which stabilizes the protein against unfolding. However, no studies have so far been conducted to explore if PG binding to AmtB has functional consequences. Here, we used an in vitro experimental assay with purified components together with molecular dynamics simulations to characterise the relation between PG binding and AmtB activity. Firstly, our results indicate that the function of Amt in archaebacteria and eubacteria may differ. Secondly, we show that PG is an essential cofactor for AmtB activity and that in the absence of PG AmtB cannot complete the full translocation cycle. Furthermore, our simulations reveal previously undiscovered PG binding sites on the intracellular side of the lipid bilayer between the AmtB subunits. The possible molecular mechanisms explaining the functional role of PG are discussed.