title: The plant plasma membrane Ca2+ pump ACA8 contains overlapping as well as physically separated autoinhibitory and calmodulin-binding domains.
abstract: In plant Ca(2+) pumps belonging to the P(2B) subfamily of P-type ATPases, the N-terminal cytoplasmic domain is responsible for pump autoinhibition. Binding of calmodulin (CaM) to this region results in pump activation but the structural basis for CaM activation is still not clear. All residues in a putative CaM-binding domain (Arg(43) to Lys(68)) were mutagenized and the resulting recombinant proteins were studied with respect to CaM binding and the activation state. The results demonstrate that (i) the binding site for CaM is overlapping with the autoinhibitory region and (ii) the autoinhibitory region comprises significantly fewer residues than the CaM-binding region. In a helical wheel projection of the CaM-binding domain, residues involved in autoinhibition cluster on one side of the helix, which is proposed to interact with an intramolecular receptor site in the pump. Residues influencing CaM negatively are situated on the other face of the helix, likely to face the cytosol, whereas residues controlling CaM binding positively are scattered throughout. We propose that early CaM recognition is mediated by the cytosolic face and that CaM subsequently competes with the intramolecular autoinhibitor in binding to the other face of the helix.
authors.affiliation: Department of Plant Biology, The Royal Veterinary and Agricultural University, Frederiksberg, Copenhagen, Denmark.
authors.forename: Lone
authors.initials: L
authors.lastname: Baekgaard
authors.forename: Laura
authors.initials: L
authors.lastname: Luoni
authors.forename: Maria Ida
authors.initials: MI
authors.lastname: De Michelis
authors.forename: Michael G
authors.initials: MG
authors.lastname: Palmgren
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000502:physiology; D001120:Arginine / Q000737:chemistry; D019921:Biotinylation; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D002147:Calmodulin / Q000737:chemistry*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013997:Time Factors
chemical_list: D029681:Arabidopsis Proteins; D002147:Calmodulin; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D001120:Arginine; D000251:Adenosine Triphosphatases; C508150:Aca8 protein, Arabidopsis; D000252:Calcium-Transporting ATPases; D008239:Lysine; D002118:Calcium