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

0477172 - MBÚ 2018 RIV US eng J - Článek v odborném periodiku
Giladi, M. - Van Dijk, L. - Refaeli, B. - Almagor, L. - Hiller, R. - Man, Petr - Forest, E. - Khananshvili, D.
Dynamic distinctions in the Na+/Ca2+ exchanger adopting the inward- and outward-facing conformational states.
Journal of Biological Chemistry. Roč. 292, č. 29 (2017), s. 12311-12323. ISSN 0021-9258. E-ISSN 1083-351X
Grant CEP: GA MŠMT(CZ) ED1.1.00/02.0109; GA MŠMT(CZ) LQ1604
Institucionální podpora: RVO:61388971
Klíčová slova: SODIUM-CALCIUM EXCHANGER * MASS-SPECTROMETRY * STRUCTURAL BASIS
Obor OECD: Biochemistry and molecular biology
Impakt faktor: 4.011, rok: 2017

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 jannaschiiNCX) 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.
Trvalý link: http://hdl.handle.net/11104/0273548