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

Gaëtan Dias Mirandela, Giulia Tamburrino, Paul A. Hoskisson, Ulrich Zachariae, Arnaud Javelle

Research output: Contribution to journalArticle

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12 Downloads (Pure)

Abstract

The movement of ammonium across biologic membranes is a fundamental process in all living organisms 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.-Mirandela, G. D., Tamburrino, G., Hoskisson, P. A., Zachariae, U., Javelle, A. The lipid environment determines the activity of the Escherichia coli ammonium transporter AmtB.

Original languageEnglish
Pages (from-to)1989-1999
Number of pages11
JournalFASEB Journal
Volume33
Issue number2
Early online date13 Sep 2018
DOIs
Publication statusPublished - 1 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 protein family
  • membrane protein-lipids interaction
  • molecular dynamics simulation
  • solid supported membrane electrophysiology

Cite this

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abstract = "The movement of ammonium across biologic membranes is a fundamental process in all living organisms 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.-Mirandela, G. D., Tamburrino, G., Hoskisson, P. A., Zachariae, U., Javelle, A. The lipid environment determines the activity of the Escherichia coli ammonium transporter AmtB.",
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The lipid environment determines the activity of the Escherichia coli ammonium transporter AmtB. / Dias Mirandela, Gaëtan; Tamburrino, Giulia; Hoskisson, Paul A.; Zachariae, Ulrich; Javelle, Arnaud.

In: FASEB Journal, Vol. 33, No. 2, 01.02.2019, 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 - Dias Mirandela, Gaëtan

AU - Tamburrino, Giulia

AU - Hoskisson, Paul A.

AU - Zachariae, Ulrich

AU - Javelle, Arnaud

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AB - The movement of ammonium across biologic membranes is a fundamental process in all living organisms 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.-Mirandela, G. D., Tamburrino, G., Hoskisson, P. A., Zachariae, U., Javelle, A. The lipid environment determines the activity of the Escherichia coli ammonium transporter AmtB.

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KW - membrane protein-lipids interaction

KW - molecular dynamics simulation

KW - solid supported membrane electrophysiology

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EP - 1999

JO - FASEB Journal

JF - FASEB Journal

SN - 0892-6638

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ER -