Dynamic distinctions in the Na + /Ca 2+ exchanger adopting the inward- and outward-facing conformational states
| Autor: | Bosmat Refaeli, Petr Man, Liat van Dijk, Moshe Giladi, Eric Forest, Daniel Khananshvili, Lior Almagor, Reuben Hiller |
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| Přispěvatelé: | Department of Physiology and Pharmacology, Tel Aviv University [Tel Aviv], the BioCeV-Institute of Microbiology, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Tel Aviv University (TAU), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA) |
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Methanococcus Biochemistry exchanger MESH: Recombinant Proteins MESH: Apoproteins MESH: Protein Conformation Calcium-binding protein calcium transport calcium-binding protein MESH: Ligands MESH: Sodium membrane protein MESH: Peptide Fragments biology MESH: Kinetics [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry Molecular Biology/Structural Biology [q-bio.BM] Chemistry MESH: Archaeal Proteins MESH: Amino Acid Substitution MESH: Deuterium Exchange Measurement transporter MESH: Calcium hydrogen exchange mass spectrometry Intracellular MESH: Models Molecular MESH: Computational Biology MESH: Mutation membrane transport 03 medical and health sciences MESH: Protein Stability Extracellular sodium-calcium exchange Molecular Biology MESH: Protein Interaction Domains and Motifs calcium Cell Biology Membrane transport MESH: Cysteine biology.organism_classification MESH: Sodium-Calcium Exchanger Crystallography Cytosol 030104 developmental biology Membrane protein MESH: Mutagenesis Insertional MESH: Binding Sites Biophysics MESH: Methanocaldococcus Cysteine MESH: Cell Membrane |
| Zdroj: | Journal of Biological Chemistry Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2017, 292 (29), pp.12311-12323. ⟨10.1074/jbc.M117.787168⟩ Journal of Biological Chemistry, 2017, 292 (29), pp.12311-12323. ⟨10.1074/jbc.M117.787168⟩ |
| ISSN: | 0021-9258 1083-351X |
| DOI: | 10.1074/jbc.M117.787168⟩ |
| Popis: | International audience; 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. |
| Databáze: | OpenAIRE |
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