1. Biochemistry and Chemical Biology
  2. Structural Biology and Molecular Biophysics
Download icon

Structure and substrate ion binding in the sodium/proton antiporter PaNhaP

  1. David Wöhlert
  2. Werner Kühlbrandt
  3. Özkan Yildiz  Is a corresponding author
  1. Max Planck Institute of Biophysics, Germany
Research Article
  • Cited 55
  • Views 2,117
  • Annotations
Cite this article as: eLife 2014;3:e03579 doi: 10.7554/eLife.03579

Abstract

Sodium/proton antiporters maintain intracellular pH and sodium levels. Detailed structures of antiporters with bound substrate ions are essential for understanding how they work. We have resolved the substrate ion in the dimeric, electroneutral sodium/proton antiporter PaNhaP from Pyrococcus abyssi at 3.2 Å, and have determined its structure in two different conformations at pH8 and pH4. The ion is coordinated by three acidic sidechains, a water molecule, a serine and a main-chain carbonyl in the unwound stretch of trans-membrane helix 5 at the deepest point of a negatively charged cytoplasmic funnel. A second narrow polar channel may facilitate proton uptake from the cytoplasm. Transport activity of PaNhaP is cooperative at pH6 but not at pH5. Cooperativity is due to pH-dependent allosteric coupling of protomers through two histidines at the dimer interface. Combined with comprehensive transport studies, the structures of PaNhaP offer unique new insights into the transport mechanism of sodium/proton antiporters.

Article and author information

Author details

  1. David Wöhlert

    Max Planck Institute of Biophysics, Frankfurt am Main, Germany
    Competing interests
    No competing interests declared.
  2. Werner Kühlbrandt

    Max Planck Institute of Biophysics, Frankfurt am Main, Germany
    Competing interests
    Werner Kühlbrandt, Reviewing editor, eLife..
  3. Özkan Yildiz

    Max Planck Institute of Biophysics, Frankfurt am Main, Germany
    For correspondence
    Oezkan.Yildiz@biophys.mpg.de
    Competing interests
    No competing interests declared.

Reviewing Editor

  1. Richard Aldrich, The University of Texas at Austin, United States

Publication history

  1. Received: June 6, 2014
  2. Accepted: November 25, 2014
  3. Accepted Manuscript published: November 26, 2014 (version 1)
  4. Accepted Manuscript updated: November 28, 2014 (version 2)
  5. Version of Record published: December 16, 2014 (version 3)

Copyright

© 2014, Wöhlert et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,117
    Page views
  • 264
    Downloads
  • 55
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Biochemistry and Chemical Biology
    Victor M Hernández-Rocamora et al.
    Research Article

    Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as β-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin binding proteins are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in their physiological membrane environment is poorly understood. Here we developed a novel FRET-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and we applied this assay with PBP1B homologues from Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii in the presence or absence of their cognate lipoprotein activator. Our assay will allow unravelling the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be used for high throughput screening for new antimicrobials.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Christopher Icke et al.
    Research Article

    Protein acylation is critical for many cellular functions across all domains of life. In bacteria, lipoproteins have important roles in virulence and are targets for the development of antimicrobials and vaccines. Bacterial lipoproteins are secreted from the cytosol via the Sec pathway and acylated on an N-terminal cysteine residue through the action of three enzymes. In Gram-negative bacteria, the Lol pathway transports lipoproteins to the outer membrane. Here we demonstrate that the Aat secretion system is a composite system sharing similarity with elements of a type I secretion systems and the Lol pathway. During secretion, the AatD subunit acylates the substrate CexE on a highly conserved N-terminal glycine residue. Mutations disrupting glycine acylation interfere with membrane incorporation and trafficking. Our data reveal CexE as the first member of a new class of glycine-acylated lipoprotein, while Aat represents a new secretion system that displays the substrate lipoprotein on the cell surface.