Na(+)/Ca(2+) 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 Ca(2+) 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.

• MeSH
• apoproteiny chemie   genetika   metabolismus   MeSH
• archeální proteiny chemie   genetika   metabolismus   MeSH
• buněčná membrána chemie   MeSH
• cystein chemie   MeSH
• interakční proteinové domény a motivy MeSH
• inzerční mutageneze MeSH
• kinetika MeSH
• konformace proteinů MeSH
• ligandy MeSH
• Methanocaldococcus metabolismus   MeSH
• molekulární modely * MeSH
• mutace MeSH
• peptidové fragmenty chemie   genetika   metabolismus   MeSH
• pumpa pro výměnu sodíku a vápníku chemie   genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• sodík metabolismus   MeSH
• stabilita proteinů MeSH
• substituce aminokyselin MeSH
• vápník metabolismus   MeSH
• vazebná místa MeSH
• vodík-deuteriová výměna MeSH
• výpočetní biologie MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

In analogy with many other proteins, Na(+)/Ca(2+) exchangers (NCX) adapt an inverted twofold symmetry of repeated structural elements, while exhibiting a functional asymmetry by stabilizing an outward-facing conformation. Here, structure-based mutant analyses of the Methanococcus jannaschii Na(+)/Ca(2+) exchanger (NCX_Mj) were performed in conjunction with HDX-MS (hydrogen/deuterium exchange mass spectrometry) to identify the structure-dynamic determinants of functional asymmetry. HDX-MS identified hallmark differences in backbone dynamics at ion-coordinating residues of apo-NCX_Mj, whereas Na(+)or Ca(2+) binding to the respective sites induced relatively small, but specific, changes in backbone dynamics. Mutant analysis identified ion-coordinating residues affecting the catalytic capacity (kcat/Km), but not the stability of the outward-facing conformation. In contrast, distinct "noncatalytic" residues (adjacent to the ion-coordinating residues) control the stability of the outward-facing conformation, but not the catalytic capacity. The helix-breaking signature sequences (GTSLPE) on the α1 and α2 repeats (at the ion-binding core) differ in their folding/unfolding dynamics, while providing asymmetric contributions to transport activities. The present data strongly support the idea that asymmetric preorganization of the ligand-free ion-pocket predefines catalytic reorganization of ion-bound residues, where secondary interactions with adjacent residues couple the alternating access. These findings provide a structure-dynamic basis for ion-coupled alternating access in NCX and similar proteins.

• MeSH
• archeální proteiny chemie   genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu MeSH
• hmotnostní spektrometrie MeSH
• interakční proteinové domény a motivy MeSH
• iontový transport MeSH
• katalytická doména MeSH
• koncentrace vodíkových iontů MeSH
• Methanocaldococcus chemie   genetika   metabolismus   MeSH
• molekulární modely MeSH
• pumpa pro výměnu sodíku a vápníku chemie   genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• sbalování proteinů MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• sodík chemie   metabolismus   MeSH
• vápník chemie   metabolismus   MeSH
• vazba proteinů MeSH
• vodík-deuteriová výměna MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH