Arginine oscillation explains Na+ independence in the substrate/product antiporter CaiT

Proc Natl Acad Sci U S A. 2013 Oct 22;110(43):17296-301. doi: 10.1073/pnas.1309071110. Epub 2013 Oct 7.

Abstract

Most secondary-active transporters transport their substrates using an electrochemical ion gradient. In contrast, the carnitine transporter (CaiT) is an ion-independent, l-carnitine/γ-butyrobetaine antiporter belonging to the betaine/carnitine/choline transporter family of secondary transporters. Recently determined crystal structures of CaiT from Escherichia coli and Proteus mirabilis revealed an inverted five-transmembrane-helix repeat similar to that in the amino acid/Na(+) symporter LeuT. The ion independence of CaiT makes it unique in this family. Here we show that mutations of arginine 262 (R262) make CaiT Na(+)-dependent. The transport activity of R262 mutants increased by 30-40% in the presence of a membrane potential, indicating substrate/Na(+) cotransport. Structural and biochemical characterization revealed that R262 plays a crucial role in substrate binding by stabilizing the partly unwound TM1' helix. Modeling CaiT from P. mirabilis in the outward-open and closed states on the corresponding structures of the related symporter BetP reveals alternating orientations of the buried R262 sidechain, which mimic sodium binding and unbinding in the Na(+)-coupled substrate symporters. We propose that a similar mechanism is operative in other Na(+)/H(+)-independent transporters, in which a positively charged amino acid replaces the cotransported cation. The oscillation of the R262 sidechain in CaiT indicates how a positive charge triggers the change between outward-open and inward-open conformations as a unifying critical step in LeuT-type transporters.

Keywords: membrane protein structure; membrane transport mechanism; secondary-active transport; sodium-dependent transport; substrate/product antiport.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antiporters / chemistry
  • Antiporters / genetics
  • Antiporters / metabolism*
  • Arginine / chemistry
  • Arginine / genetics
  • Arginine / metabolism*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Binding Sites / genetics
  • Biological Transport / genetics
  • Carnitine / chemistry
  • Carnitine / metabolism
  • Crystallography, X-Ray
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Proteus mirabilis / chemistry
  • Proteus mirabilis / genetics
  • Proteus mirabilis / metabolism*
  • Sequence Homology, Amino Acid
  • Sodium / metabolism*
  • Substrate Specificity

Substances

  • Antiporters
  • Bacterial Proteins
  • Arginine
  • Sodium
  • Carnitine

Associated data

  • PDB/4M8J