A New Conformation in Sarcoplasmic Reticulum Calcium Pump and Plasma Membrane Ca2+ Pumps Revealed by a Photoactivatable Phospholipidic Probe.

IRENE MANGIALAVORI; ANA MARÍA VILLAMIL GIRALDO; CRISTINA MARINO; MARIELA FERREIRA GOMES; ARIEL CARIDE; JUAN PABLO ROSSI

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Año: 2009 vol. 284 p. 4823 - 4828

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 1-palmitoyl-2-[9-[2_-[125I]iodo-4-(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+  + 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.