title: Asymmetric Preorganization of Inverted Pair Residues in the Sodium-Calcium Exchanger.
abstract: 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.
authors.affiliation: Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel.
authors.forename: Moshe
authors.initials: M
authors.lastname: Giladi
authors.affiliation: Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel.
authors.forename: Lior
authors.initials: L
authors.lastname: Almagor
authors.affiliation: Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel.
authors.forename: Liat
authors.initials: L
authors.lastname: van Dijk
authors.affiliation: Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel.
authors.forename: Reuben
authors.initials: R
authors.lastname: Hiller
authors.affiliation: Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Man
authors.affiliation: Univ. Grenoble Alpes, IBS, F-38044 Grenoble, France.|CNRS, IBS, F-38044 Grenoble, France.|CEA, IBS, F-38044 Grenoble, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Forest
authors.affiliation: Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel.
authors.forename: Daniel
authors.initials: D
authors.lastname: Khananshvili
mesh_terms: D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D041961:Deuterium Exchange Measurement; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D013058:Mass Spectrometry; D063747:Methanocaldococcus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000737:chemistry* / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D019843:Archaeal Proteins; D011994:Recombinant Proteins; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D002118:Calcium