Structural and functional differences in cytosolic and membrane domains in sarcoplasmic reticulum calcium pump (SERCA) and plasma membrane calcium pump (PMCA)

SERCA and PMCA belong to the P-ATPases family. SERCA structure and function has been widely characterized. PMCA has a relatively high sequence identity with SERCA but its structure is less known. Although both proteins pump Ca 2+ out of the cytoplasm of cells, their kinetic properties are very different. As an attempt to understand this on the basis of differences in their structure, we employed fluoride complexes with beryllium, aluminium or magnesium, which are proposed to stabilize different analogues of the phosphorylated intermediates in P-ATPases although they have never been tested in PMCA. To study the proteins structure we employed the photoactivatable probe 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID) which labels the transmembrane domains of these proteins and the fluorescent probe 2',3'-O-(2,4,6-Trinitrophenyl)adenosine-5'-triphosphate (TNP-ATP) which binds to the nucleotide binding domain of both pumps. BeF 3- and AlF 4- inhibit PMCA activity at micromolar concentrations, whereas millimolar concentrations of Mg 2+ and F - are required. TID labeling in different conformations of SERCA correlates well with the protein surface exposed to the bilayer calculated from crystallographic models of the protein, with less labeling in the presence of calcium. Irrespectively of the presence of metal-fluoride complexes, TID labeling of PMCA only decreases when the pump is incubated with calcium and calmodulin, indicating a lower exposure of the transmembrane regions under these conditions. Upon addition of metal-fluoride complexes, TNP-ATP bound to SERCA increases its quantum yield, whereas when is bound to PMCA its quantum yield decreases by a half. Our results indicate that calmodulin binding to PMCA allows conformational changes in the transmembrane region similar to those observed in SERCA in presence of calcium, but the nucleotide biding domain behaves very differently when these proteins are in a phosphorylated-like state.