TY - JOUR
T1 - The lipid environment determines the activity of the Escherichia coli ammonium transporter AmtB
AU - Mirandela, Gaetan Dias
AU - Tamburrino, Giulia
AU - Hoskisson, Paul A.
AU - Zachariae, Ulrich
AU - Javelle, Arnaud
N1 - G.D.M. was supported by a Ph.D. Studentship from the University of Strathclyde; A.J. was supported by a Chancellor’s Fellowship from the University of Strathclyde; and G.T. and U.Z. acknowledge funding from the Scottish Universities’ Physics Alliance (SUPA). A.J. acknowledges the support of Tenovus Scotland (Project S17-07), and P.A.H. acknowledges the support of the Natural Environment Research Council (Grant NE/M001415/1).
PY - 2019/2
Y1 - 2019/2
N2 - The movement of ammonium across biologic membranes is a fundamental process in all living organ-isms and is mediated by the ubiquitous ammonium transporter/methylammonium permease/rhesus protein (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 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG). Upon POPG 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 our knowledge, to explore whether POPG binding to AmtB has functional consequences. Here, we used an in vitro experimental assay with purified components, together with molecular dynamics simulations, to characterize the relation between POPG binding and AmtB activity. We show that the AmtB activity is electrogenic. Our results indicate that the activity, at the molecular level, of Amt in archaebacteria and eubacteria may differ. We also show that POPG is an important cofactor for AmtB activity and that, in the absence of POPG, AmtB cannot complete the full translocation cycle. Furthermore, our simulations reveal previously undiscovered POPG binding sites on the intracellular side of the lipid bilayer between the AmtB subunits. Possible molecular mechanisms explaining the functional role of POPG are discussed.
AB - The movement of ammonium across biologic membranes is a fundamental process in all living organ-isms and is mediated by the ubiquitous ammonium transporter/methylammonium permease/rhesus protein (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 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG). Upon POPG 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 our knowledge, to explore whether POPG binding to AmtB has functional consequences. Here, we used an in vitro experimental assay with purified components, together with molecular dynamics simulations, to characterize the relation between POPG binding and AmtB activity. We show that the AmtB activity is electrogenic. Our results indicate that the activity, at the molecular level, of Amt in archaebacteria and eubacteria may differ. We also show that POPG is an important cofactor for AmtB activity and that, in the absence of POPG, AmtB cannot complete the full translocation cycle. Furthermore, our simulations reveal previously undiscovered POPG binding sites on the intracellular side of the lipid bilayer between the AmtB subunits. Possible molecular mechanisms explaining the functional role of POPG are discussed.
KW - Amt/Mep/Rh
KW - Molecular dynamics simulation
KW - Protein-lipids interaction
KW - SSME
UR - http://www.scopus.com/inward/record.url?scp=85060049269&partnerID=8YFLogxK
U2 - 10.1096/fj.201800782R
DO - 10.1096/fj.201800782R
M3 - Article
C2 - 30211659
SN - 0892-6638
VL - 33
SP - 1989
EP - 1999
JO - FASEB Journal
JF - FASEB Journal
IS - 2
ER -