Involvement of the L6–7 Loop in SERCA1a Ca2+-ATPase Activation by Ca2+ (or Sr2+) and ATP

Abstract

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.Muscle relaxation occurs by of the removal of calcium from the cytosol and its accumulation into sarcoplasmic reticulum (SR) 1 through an active transport that consumes ATP (1, 2). In fast twitch muscle this transport is achieved by the SERCA1a isoform of Ca 2ϩ -ATPase (3), with a stoichiometry of two calcium ions transported per ATP molecule hydrolyzed (see reviews in Refs. 4 and 5). SR Ca 2ϩ -ATPase belongs to the family of P-type ATPases, which transport cations such as H ϩ , Na ϩ , K ϩ , or Ca 2ϩ across the membrane and share a common mechanism that involves autophosphorylation of the protein (see review in Ref. 6): transport is achieved through a reversible cycle, during which the ATPase is thought to sequentially adopt two main conformations (7, 8), E1 and E2, with a high and low affinity for the transported ion, and the overall cycle corresponds to completion of the following scheme: E2 3 Ca 2 E1 3 Ca 2 E1 ϳ P 3 E2P 3 E2 (for review, see Refs. 9 and 10). Much effort has gone into determining the structural organization of SR Ca 2ϩ -ATPase from primary structure predictions (11), electron microscopy (12), and three-dimensional crystallogenesis (13-15) (for a recent review see Ref. 16). Finally, the structure has been solved at the atomic level, first in a Ca 2 E1 state (14) and then in a thapsigargin-stabilized E2 state (15). T...

Crystal Structure of D351A and P312A Mutant Forms of the Mammalian Sarcoplasmic Reticulum Ca2+-ATPase Reveals Key Events in Phosphorylation and Ca2+ Release

“…4). From a methodological point of view it is of interest that we found that under appropriate conditions (40% glycerol added to Ca 2ϩ -ATPase solubilized by 1 mg/ml DDM) information could be obtained directly from the detergent-solubilized state, obviating the need to go through the complete reconstitution procedure used previously (15,36). In addition to confirming the reliability of the previous measurement of an ATP affinity in the nanomolar Ϫ and AMPPCP are also apparent in the difference map albeit at lower (3.0 ) contour level, not shown.)…”

“…The analysis of the functional consequences of mutations has reached a level where it encompasses rapid kinetic quench-flow studies of the partial reaction steps (28) and most recently also biophysical measurements of conformational transitions by fluorescence changes, made feasible by large scale expression in yeast (15,29). A number of mutations appear to accelerate or block partial reactions in the functional cycle and displace E1-E2 and/or E1P-E2P equilibria.…”

Cdc50p Plays a Vital Role in the ATPase Reaction Cycle of the Putative Aminophospholipid Transporter Drs2p

“…Phosphorylation Assay-Phosphorylation from [␥-32 P]ATP was performed as described (29,30) on purified H 2 -Drs2p preincubated with 1 mg/ml dioleoylphosphatidyl choline/dioleoylphosphatidic acid (9/1) in MOPS buffer (50 mM MOPSTris, pH 7.0, 100 mM KCl, 5 mM MgCl 2 , 0.05% digitonin) for 1 h at 4°C under gentle agitation. One g/ml H 2 -Drs2p (ϳ0.1 g/reaction) in MOPS buffer supplemented with 6 units/ml pyruvate kinase was incubated at 4°C for 50 supplemental Table S1.…”

Functional Consequences of Alterations to Thr247, Pro248, Glu340, Asp813, Arg819, and Arg822 at the Interfaces between Domain P, M3, and L6-7 of Sarcoplasmic Reticulum Ca2+-ATPase

“…Noting that crystal structures represent "frozen" conformations characterized by exceptional stability under certain nonphysiological conditions, it is important to ask the question whether the hydrogen bond interactions apparent from the crystal structures also occur in the native enzyme during the transport cycle, and whether they are functionally important. (13)(14)(15)(16)(17) and of insensitivity to regulation by phospholamban in point mutant D813A (18). In preliminary work (19), we have demonstrated that point mutant D813L exhibits a much larger phosphorylation overshoot than the wild type, upon reaction of the Ca 2ϩ -bound enzyme with ATP.…”