The lipid environment determines the activity of the Escherichia coli ammonium transporter AmtB

Gaetan Dias Mirandela, Giulia Tamburrino, Paul A. Hoskisson, Ulrich Zachariae, Arnaud Javelle (Lead / Corresponding author)

Research output: Contribution to journalArticle

2 Citations (Scopus)
43 Downloads (Pure)

Abstract

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.
Original languageEnglish
Pages (from-to)1989-1999
Number of pages11
JournalFASEB Journal
Volume33
Issue number2
Early online date13 Sep 2018
DOIs
Publication statusPublished - Feb 2019

Fingerprint

Phosphatidylglycerols
Ammonium Compounds
Escherichia coli
Lipids
Protein Unfolding
Lipid bilayers
Membrane Transport Proteins
Archaea
Lipid Bilayers
Molecular Dynamics Simulation
Structural analysis
Mass spectrometry
Molecular dynamics
Assays
Mass Spectrometry
Proteins
Binding Sites
Membranes
Bacteria
Computer simulation

Keywords

  • Amt/Mep/Rh
  • Molecular dynamics simulation
  • Protein-lipids interaction
  • SSME

Cite this

Mirandela, Gaetan Dias ; Tamburrino, Giulia ; Hoskisson, Paul A. ; Zachariae, Ulrich ; Javelle, Arnaud. / The lipid environment determines the activity of the Escherichia coli ammonium transporter AmtB. In: FASEB Journal. 2019 ; Vol. 33, No. 2. pp. 1989-1999.
@article{57484492b9644361ad6cc2b64e7e922f,
title = "The lipid environment determines the activity of the Escherichia coli ammonium transporter AmtB",
abstract = "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.",
keywords = "Amt/Mep/Rh, Molecular dynamics simulation, Protein-lipids interaction, SSME",
author = "Mirandela, {Gaetan Dias} and Giulia Tamburrino and Hoskisson, {Paul A.} and Ulrich Zachariae and Arnaud Javelle",
note = "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).",
year = "2019",
month = "2",
doi = "10.1096/fj.201800782R",
language = "English",
volume = "33",
pages = "1989--1999",
journal = "FASEB Journal",
issn = "0892-6638",
publisher = "Federation of American Society of Experimental Biology",
number = "2",

}

The lipid environment determines the activity of the Escherichia coli ammonium transporter AmtB. / Mirandela, Gaetan Dias; Tamburrino, Giulia; Hoskisson, Paul A.; Zachariae, Ulrich; Javelle, Arnaud (Lead / Corresponding author).

In: FASEB Journal, Vol. 33, No. 2, 02.2019, p. 1989-1999.

Research output: Contribution to journalArticle

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

VL - 33

SP - 1989

EP - 1999

JO - FASEB Journal

JF - FASEB Journal

SN - 0892-6638

IS - 2

ER -