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Dynamic distinctions in the Na + /Ca 2+ exchanger adopting the inward- and outward-facing conformational states

Abstract : 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.
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https://hal.archives-ouvertes.fr/hal-01647025
Contributor : Frank Thomas <>
Submitted on : Friday, November 24, 2017 - 9:54:22 AM
Last modification on : Tuesday, November 3, 2020 - 3:14:00 PM

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Moshe Giladi, Liat van Dijk, Bosmat Refaeli, Lior Almagor, Reuben Hiller, et al.. Dynamic distinctions in the Na + /Ca 2+ exchanger adopting the inward- and outward-facing conformational states. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2017, 292 (29), pp.12311 - 12323. ⟨10.1074/jbc.M117.787168⟩. ⟨hal-01647025⟩

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