Metal fluoride effects on the Plasma Membrane Ca2+ ATPase: Characterization of the fluoride-stabilized phosphoenzyme analogues

.ROSSI, JUAN PABLO F.C.; SAFFIOTI NICOLÁS ANDRÉS1; RIESCO ANA SOL1; DE SAUTU MARILINA1; ROSSI ROLANDO1; BERLIN JOSHUA2; FERREIRA-GOMES MARIELA1; ROSSI JUAN PABLO1; MANGIALAVORI IRENE1

LA PLATA

Congreso; XLVII Reunión Anual de la Sociedad de Biofísica Argentina. La Plata; 2018

SOCIEDAD ARGENTINA DE BIOFÍSICA

Metal fluorideeffects on the Plasma Membrane Ca2+ ATPase: Characterization of thefluoride-stabilized phosphoenzyme analogues. Saffioti Nicolás Andrés1; Riesco Ana Sol1;de Sautu Marilina1; Rossi Rolando1; Berlin Joshua2;Ferreira-Gomes Mariela1; Rossi Juan Pablo1; MangialavoriIrene1 1Instituto de Química yFisicoquímica Biológicas. ?Prof.Paladini?. Departamento de Química Biológica,Facultad de Farmacia y Bioquímica. Universidad deBuenos Aires.2Departmentof Pharmacology and Physiology, New Jersey. Medical School, Rutgers University,Newark, NJ, USA The Plasma Membrane Ca2+ Pump (PMCA) is one of the mostimportant participants in cytoplasmic Ca2+ regulation, and belongsto the P-type ATPases family (P-ATPases). The reaction cycle of this group isexplained by the Albers and Post model, which postulates that these proteinsexist in two main conformations E1 and E2 that can bephosphorylated forming the intermediates E1-P and E2-P.Recently, the first structural model of PMCA was obtained bycrio-electronmicroscopy1. In the context of progresses in the studyof PMCA structure, new methods to stabilize this protein in differentconformations are necessary in order to investigate the relationship betweenits structure and mechanism of function and regulation.In this workwe studied the effects of fluoride complexes of aluminium (AlFx),beryllium (BeFx) and magnesium (MgFx) on PMCA by means ofmeasurement of enzyme activity, and employing fluorescent or hydrophobicphotoactivatable probes. These complexes stabilize different states of thephosphorylated intermediates in other P-ATPases, but they have never beentested on PMCA. Our results show that the three complexes behaved as slowreversible inhibitors of Ca2+-ATPase and phosphatase activities bypreventing phosphorylation from ATP. The inhibition was not competitive with Ca2+ in contrast withresults observed in other P-ATPases. The affinitiesfor AlFx, BeFx and MgFx increased slightlywhen PMCA was activated by calmodulin. On the other hand, the water content inthe nucleotide binding site increased as a result of the conformational changeproduced by fluoride complexes binding. This effect occurred also when PMCA isphosphorylated from ATP, which allowed the measurement of the concentration ofthe phosphorylated intermediate in real time. In summary, our results show thatthese fluoride complexes are a useful to reveal the properties of differentphosphorylated intermediates that are involved in the mechanism of hydrolysisof ATP by the PMCA. 1 Gong, D., Chi, X., Ren, K., Huang, G., Zhou, G., Yan,N., Lei, J., Zhou Q. (2018) Nature Communications. 36239 1. This work was supported by Agencia Nacional de Promoción Científica yTecnológica PICT 2014 0065, Consejo Nacional de Investigaciones Científicas yTécnicas PIP 11220150100250CO, and Universidad de Buenos Aires Ciencia yTécnica grant 2014-2017: 20020130100254B