title: A bimodular mechanism of calcium control in eukaryotes.
abstract: Calcium ions (Ca(2+)) have an important role as secondary messengers in numerous signal transduction processes, and cells invest much energy in controlling and maintaining a steep gradient between intracellular (∼0.1-micromolar) and extracellular (∼2-millimolar) Ca(2+) concentrations. Calmodulin-stimulated calcium pumps, which include the plasma-membrane Ca(2+)-ATPases (PMCAs), are key regulators of intracellular Ca(2+) in eukaryotes. They contain a unique amino- or carboxy-terminal regulatory domain responsible for autoinhibition, and binding of calcium-loaded calmodulin to this domain releases autoinhibition and activates the pump. However, the structural basis for the activation mechanism is unknown and a key remaining question is how calmodulin-mediated PMCA regulation can cover both basal Ca(2+) levels in the nanomolar range as well as micromolar-range Ca(2+) transients generated by cell stimulation. Here we present an integrated study combining the determination of the high-resolution crystal structure of a PMCA regulatory-domain/calmodulin complex with in vivo characterization and biochemical, biophysical and bioinformatics data that provide mechanistic insights into a two-step PMCA activation mechanism mediated by calcium-loaded calmodulin. The structure shows the entire PMCA regulatory domain and reveals an unexpected 2:1 stoichiometry with two calcium-loaded calmodulin molecules binding to different sites on a long helix. A multifaceted characterization of the role of both sites leads to a general structural model for calmodulin-mediated regulation of PMCAs that allows stringent, highly responsive control of intracellular calcium in eukaryotes, making it possible to maintain a stable, basal level at a threshold Ca(2+) concentration, where steep activation occurs.
authors.affiliation: Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Aarhus University, Gustav Wieds Vej 10c, DK-8000 Aarhus C, Denmark.
authors.forename: Henning
authors.initials: H
authors.lastname: Tidow
authors.forename: Lisbeth R
authors.initials: LR
authors.lastname: Poulsen
authors.forename: Antonina
authors.initials: A
authors.lastname: Andreeva
authors.forename: Michael
authors.initials: M
authors.lastname: Knudsen
authors.forename: Kim L
authors.initials: KL
authors.lastname: Hein
authors.forename: Carsten
authors.initials: C
authors.lastname: Wiuf
authors.forename: Michael G
authors.initials: MG
authors.lastname: Palmgren
authors.forename: Poul
authors.initials: P
authors.lastname: Nissen
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D056890:Eukaryota / Q000378:metabolism*; D042541:Intracellular Space / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
chemical_list: D029681:Arabidopsis Proteins; D002147:Calmodulin; C508150:Aca8 protein, Arabidopsis; D000252:Calcium-Transporting ATPases; D002118:Calcium