Structural and Functional Differences between Sarcoplasmic Reticulum Calcium Pump (SERCA) and Plasma Membrane Calcium Pump (PMCA) reaction Cycle Intermediates

SAFFIOTI, NICOLÁS; DE SAUTU, MARILINA; MANGIALAVORI IRENE; FERREIRA GOMES, MARIELA; ROSSI ROLANDO; BERLIN, JOSHUA; ROSSI JPFC

New Orleans, Louisiana

Congreso; 61st Annual Meeting of Biophysical Society.; 2017

Biophysical Society

SERCA and PMCA belong to the P-ATPases family. SERCAs structure andfunction have been widely characterized whereas PMCA has a relativelyhigh sequence identity with SERCA but its structure is less known. Althoughboth proteins pump Ca2þ out of the cytoplasm of cells, their kinetic propertiesare 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 intermediatesin P-ATPases, although they have never been tested on PMCA. To study theprotein structure we employed the hydrophobic photoactivatable probe 3-(tri-fluoromethyl)-3-(m-iodophenyl)diazirine (TID) which labels the transmembranedomains of these proteins and the fluorescent probe 2?,3?-O-(2,4,6-Trinitrophenyl)adenosine-5?-triphosphate (TNP-ATP) which binds to thenucleotide binding-domain of both pumps. Results show that BeF3- and AlF4-inhibit PMCA activity at micromolar concentrations, whereas millimolarconcentrations of Mg2+ and F- are required to achieve that effect. TID labelingin different conformations of SERCA correlates well with the protein surfaceexposed to the bilayer calculated from crystallographic models, with less labelingin the presence of calcium. Irrespectively of the presence of metal-fluoridecomplexes, TID labeling of PMCA only decreases when the pump is incubatedwith calcium and calmodulin, indicating a lower exposure of the transmembraneregion. When incubated with metal-fluoride complexes, TNP-ATP increasesits 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 phosphorylated likestate.With grants from ANPCYT, CONICET and UBACYTWith grants from ANPCYT, CONICET and UBACYT