A New Conformation in SERCA and PMCA Ca2+-Pumps Revealed by a Photoactivatable Phospholipidic Probe

MANGIALAVORI IRENE; VILLAMIL GIRALDO A.M; CARIDE, A.J.; ROSSI, J.P.F.C.

JOURNAL OF BIOLOGICAL CHEMISTRY

American Society for Biochemistry and Molecular Biology

Lugar: New York; Año: 2008

0021-9258

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 [125I]TID-PC/16 under different conditions. We determined that: (1) Incorporation of [125I]TID-PC/16 to SERCA decreases 25% when labeling is performed in the presence of Ca2+ These 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 which 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 2 different E1 conformations: one that is auto-inhibited and is in contact with a higher amount of lipids (Ca2++C28 for SERCA, 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.