Involvement of the L6-7 loop in SERCA1a Ca2+-ATPase activation by Ca2+ (or Sr2+) and ATP.
Wild-type (WT) and the double mutant D813A,D818A (ADA) of the L6-7 loop of SERCA1a were expressed in yeast, purified, and reconstituted into lipids. This allowed us to functionally study these ATPases by both kinetic and spectroscopic means, and to solve previous discrepancies in the published literature about both experimental facts and interpretation concerning the role of this loop in P-type ATPases. We show that in a solubilized state, the ADA mutant experiences a dramatic decrease of its calcium-dependent ATPase activity. On the contrary, reconstituted in a lipid environment, it displays an almost unaltered maximal calcium-dependent ATPase activity at high (millimolar) ATP, with an apparent affinity for Ca(2+) altered only moderately (3-fold). In the absence of ATP, the true affinity of ADA for Ca(2+) is, however, more significantly reduced (20-30-fold) compared with WT, as judged from intrinsic (Trp) or extrinsic (fluorescence isothiocyanate) fluorescence experiments. At low ATP, transient kinetics experiments reveal an overshoot in the ADA phosphorylation level primarily arising from the slowing down of the transition between the nonphosphorylated "E2" and "Ca(2)E1" forms of ADA. At high ATP, this slowing down is only partially compensated for, as ADA turnover remains more sensitive to orthovanadate than WT turnover. ADA ATPase also proved to have a reduced affinity for ATP in studies performed under equilibrium conditions in the absence of Ca(2+), highlighting the long range interactions between L6-7 and the nucleotide-binding site. We propose that these mutations in L6-7 could affect protonation-dependent winding and unwinding events in the nearby M6 transmembrane segment.
Lenoir
Guillaume
G
Unité de Recherche Associée 2096, the Centre National de la Recherche Scientifique and Section de Biophysique des Fonctions Membranaires, Département de Biologie Joliot Curie, CEA Saclay, 91191 Gif-sur-Yvette Cedex, France.
Picard
Martin
M
Møller
Jesper V
JV
le Maire
Marc
M
Champeil
Philippe
P
Falson
Pierre
P
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry*; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D018482:Muscle, Skeletal / Q000378:metabolism; D009154:Mutation; D010710:Phosphates / Q000737:chemistry; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D003902:Detergents; D008055:Lipids; D010710:Phosphates; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium