A new type of Na(+)-driven ATP synthase membrane rotor with a two-carboxylate ion-coupling motif

PLoS Biol. 2013;11(6):e1001596. doi: 10.1371/journal.pbio.1001596. Epub 2013 Jun 25.

Abstract

The anaerobic bacterium Fusobacterium nucleatum uses glutamate decarboxylation to generate a transmembrane gradient of Na⁺. Here, we demonstrate that this ion-motive force is directly coupled to ATP synthesis, via an F₁F₀-ATP synthase with a novel Na⁺ recognition motif, shared by other human pathogens. Molecular modeling and free-energy simulations of the rotary element of the enzyme, the c-ring, indicate Na⁺ specificity in physiological settings. Consistently, activity measurements showed Na⁺ stimulation of the enzyme, either membrane-embedded or isolated, and ATP synthesis was sensitive to the Na⁺ ionophore monensin. Furthermore, Na⁺ has a protective effect against inhibitors targeting the ion-binding sites, both in the complete ATP synthase and the isolated c-ring. Definitive evidence of Na⁺ coupling is provided by two identical crystal structures of the c₁₁ ring, solved by X-ray crystallography at 2.2 and 2.6 Å resolution, at pH 5.3 and 8.7, respectively. Na⁺ ions occupy all binding sites, each coordinated by four amino acids and a water molecule. Intriguingly, two carboxylates instead of one mediate ion binding. Simulations and experiments demonstrate that this motif implies that a proton is concurrently bound to all sites, although Na⁺ alone drives the rotary mechanism. The structure thus reveals a new mode of ion coupling in ATP synthases and provides a basis for drug-design efforts against this opportunistic pathogen.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Binding Sites
  • Biocatalysis / drug effects
  • Biological Transport / drug effects
  • Cell Membrane / drug effects
  • Cell Membrane / enzymology*
  • Crystallography, X-Ray
  • Detergents / pharmacology
  • Dicyclohexylcarbodiimide
  • Fusobacterium nucleatum / drug effects
  • Fusobacterium nucleatum / enzymology*
  • Fusobacterium nucleatum / growth & development
  • Humans
  • Hydrogen-Ion Concentration
  • Ionophores / pharmacology
  • Ions
  • Kinetics
  • Lithium / metabolism
  • Mitochondrial Proton-Translocating ATPases / antagonists & inhibitors
  • Mitochondrial Proton-Translocating ATPases / chemistry*
  • Mitochondrial Proton-Translocating ATPases / isolation & purification
  • Mitochondrial Proton-Translocating ATPases / metabolism*
  • Molecular Dynamics Simulation
  • Protons
  • Sodium / metabolism*
  • Substrate Specificity / drug effects

Substances

  • Detergents
  • Ionophores
  • Ions
  • Protons
  • Dicyclohexylcarbodiimide
  • Lithium
  • Sodium
  • F1F0-ATP synthase
  • Mitochondrial Proton-Translocating ATPases

Grant support

This work was financially supported by the German Research Foundation (DFG), via the Collaborative Research Center SFB 807 (TM) and the Cluster of Excellence ‘Macromolecular Complexes’ EXC 115 (JDFG and TM), and by a Marsden Grant from the Royal Society of New Zealand (GMC and MIC). Computing resources were in part provided by the Leibniz and Jülich Supercomputing Centers. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.