Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8.
Plasma-membrane Ca(2+)-ATPases (PMCAs) are calcium pumps that expel Ca(2+) from eukaryotic cells to maintain overall Ca(2+) homoeostasis and to provide local control of intracellular Ca(2+) signalling. They are of major physiological importance, with different isoforms being essential, for example, for presynaptic and postsynaptic Ca(2+) regulation in neurons, feedback signalling in the heart and sperm motility. In the resting state, PMCAs are autoinhibited by binding of their C-terminal (in mammals) or N-terminal (in plants) tail to two major intracellular loops. Activation requires the binding of calcium-bound calmodulin (Ca(2+)-CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca(2+)-ATPase ACA8 from Arabidopsis thaliana has been crystallized. The crystals belonged to space group C2, with unit-cell parameters a = 176.8, b = 70.0, c = 69.8 A, beta = 113.2 degrees. A complete data set was collected to 3.0 A resolution and structure determination is in progress in order to elucidate the mechanism of PMCA activation by calmodulin.
Tidow
Henning
H
Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Aarhus University, Gustav Wieds Vej 10c, DK-8000 Aarhus C, Denmark. tidow@bioxray.au.dk
Hein
Kim L
KL
Baekgaard
Lone
L
Palmgren
Michael G
MG
Nissen
Poul
P
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D015870:Gene Expression; D008969:Molecular Sequence Data; D011485:Protein Binding
D029681:Arabidopsis Proteins; D002147:Calmodulin; C508150:Aca8 protein, Arabidopsis; D000252:Calcium-Transporting ATPases