A new conformation in sarcoplasmic reticulum calcium pump and plasma membrane Ca2+ pumps revealed by a photoactivatable phospholipidic probe

Abstract

The purpose of this work was to obtain structural information about conformational changes in the membrane region of the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca2+ pumps. We have assessed changes in the overall exposure of these proteins to surrounding lipids by quantifying the extent of protein labeling by a photoactivatable phosphatidylcholine analog 1-palmitoyl-2-[9-[2_p-[125I]iodo-4_p- (trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]- sn-glycero-3-phosphocholine ([125I]TID- PC/16) under different conditions. We determined the following. 1) Incorporation of [125I]TID-PC/16 to SERCA decreases 25% when labeling is performed in the presence of Ca2+. This decrease in labeling matches qualitatively the decrease in transmembrane surface exposed to the solvent calculated from crystallographic data for SERCA structures. 2) Labeling of PMCA incubated with Ca2+ and calmodulin decreases by approximately the same amount. However, incubation with Ca2+ alone increases labeling by more than 50%. Addition of C28, a peptide that prevents activation of PMCA by calmodulin, yields similar results. C28 has also been shown to inhibit ATPase SERCA activity. Interestingly, incubation of SERCA with C28 also increases [125I]TID-PC/16 incorporation to the protein. These results suggest that in both proteins there are two different E1 conformations as follows: one that is auto-inhibited and is in contact with a higher amount of lipids (Ca2+ a C28 for SERCA and Ca2+ alone for PMCA), and one in which the enzyme is fully active (Ca2+ for SERCA and Ca2+-calmodulin for PMCA) and that exhibits a more compact transmembrane arrangement. These results are the first evidence that there is an autoinhibited conformation in these P-type ATPases, which involves both the cytoplasmic regions and the transmembrane segments. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.