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.

BioCeV Institute of Microbiology Academy of Sciences of the Czech Republic CZ 14220 Prague Czech Republic; Faculty of Science Charles University CZ 14220 Prague Czech Republic

Department of Physiology and Pharmacology Sackler School of Medicine Tel Aviv University Ramat Aviv 69978 Israel

Department of Physiology and Pharmacology Sackler School of Medicine Tel Aviv University Ramat Aviv 69978 Israel; Tel Aviv Sourasky Medical Center Tel Aviv 39040 Israel

University of Grenoble Alpes Institut de Biologie Structurale F 38044 Grenoble France; CNRS IBS F 38044 Grenoble France; Commissariat à l'Energie Atomique IBS F 38044 Grenoble France

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Structural dynamics of Na+ and Ca2+ interactions with full-size mammalian NCX

Hydrogen/Deuterium Exchange Mass Spectrometry of Heme-Based Oxygen Sensor Proteins

MS-Based Approaches Enable the Structural Characterization of Transcription Factor/DNA Response Element Complex

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PDB
5HXH, 5HXE, 5HXR