Dynamic distinctions in the Na+/Ca2+ exchanger adopting the inward- and outward-facing conformational states

J Biol Chem. 2017 Jul 21;292(29):12311-12323. doi: 10.1074/jbc.M117.787168. Epub 2017 Jun 1.


Na+/Ca2+ exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj (Methanococcus jannaschii NCX) system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na+ or Ca2+ specifically modify HDX patterns, revealing that the ion-bound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity (Km ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations.

Keywords: calcium; calcium transport; calcium-binding protein; exchanger; hydrogen exchange mass spectrometry; membrane protein; membrane transport; sodium-calcium exchange; transporter.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Apoproteins / chemistry
  • Apoproteins / genetics
  • Apoproteins / metabolism
  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism
  • Binding Sites
  • Calcium / metabolism*
  • Cell Membrane / chemistry*
  • Computational Biology
  • Cysteine / chemistry
  • Deuterium Exchange Measurement
  • Kinetics
  • Ligands
  • Methanocaldococcus / metabolism*
  • Models, Molecular*
  • Mutagenesis, Insertional
  • Mutation
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Conformation
  • Protein Interaction Domains and Motifs
  • Protein Stability
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sodium / metabolism*
  • Sodium-Calcium Exchanger / chemistry*
  • Sodium-Calcium Exchanger / genetics
  • Sodium-Calcium Exchanger / metabolism


  • Apoproteins
  • Archaeal Proteins
  • Ligands
  • Peptide Fragments
  • Recombinant Proteins
  • Sodium-Calcium Exchanger
  • Sodium
  • Cysteine
  • Calcium

Associated data

  • PDB/5HXH
  • PDB/5HXE
  • PDB/5HXR