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

SAFFIOTI NA; MANGIALAVORI IC; FERREIRA GOMES MS; ROSSI JPFC; BERLIN JR; DE SAUTU M; ROSSI RC

New Orleans, Luisiana

Congreso; 61th Annual Meeting of the Biophysical Society; 2017

Biophysical Society

SERCA and PMCA belong to the P-ATPases family. SERCA's structure and function have been widely characterized whereas PMCA has a relatively high sequence identity with SERCA but its structure is less known. Although both proteins pump Ca2+ out of the cytoplasm of cells, their kinetic properties are very different. To understand this on the basis of differences in their structure, we employed fluoride complexes with beryllium, aluminium or magnesium, which stabilize different analogues of the phosphorylated intermediates in P-ATPases, although they have never been tested on PMCA. To study the protein structure we employed the hydrophobic 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. Results show that BeF3- and AlF4- inhibit PMCA activity at micromolar concentrations, whereas millimolar concentrations of Mg2+ and F- are required to achieve that effect. TID labeling in different conformations of SERCA correlates well with the protein surface exposed to the bilayer calculated from crystallographic models, 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 region. When incubated with metal-fluoride complexes, TNP-ATP increases its quantum yield when it is bound to SERCA but decreases by ahalf when it is bound to PMCA. Our results indicate that calmodulin bindingto PMCA allows conformational changes in the transmembrane region similarto those observed in SERCA in presence of calcium, but the nucleotide bidingdomain behaves very differently when these proteins are in a phosphorylatedlikestate.