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

SAFFIOTI, NICOLÁS A.; BERLIN, JOSHUA; FERREIRA-GOMES, MARIELA S.; DE SAUTU, MARILINA; RIESCO, ANA SOL; ROSSI JUAN PABLO; ROSSI ROLANDO; MANGIALAVORI IRENE C.

La Plata

Congreso; XLVII reunión anual de la Sociedad Argentina de Biofísica; 2018

Sociedad Aregntina de Biofísica

The Plasma Membrane Ca2+ Pump (PMCA) is one of the most important participants in cytoplasmic Ca2+ regulation, and belongs to the P-type ATPases family (P-ATPases). The Albers and Post model postulates that P-ATPases exist in two main conformations E1 and E2 that can be phosphorylated forming E1-P and E2-P. Recently, the first structural model of PMCA was obtained by crio-electronmicroscopy1. In the context of progresses in the study of PMCA structure, new methods to stabilize this protein in different conformations are necessary in order to investigate the relationship between its structure and function.In this work we studied the effects of fluoride complexes of aluminium (AlFx), beryllium (BeFx) and magnesium (MgFx) on PMCA by means of measurement of enzyme activity, and employing fluorescent or hydrophobic photoactivatable probes. These complexes stabilize different states of the phosphorylated intermediates in other P-ATPases, but they have never been tested on PMCA. Our results show that the three complexes behaved as slow reversible inhibitors of Ca2+-ATPase and phosphatase activities by preventing phosphorylation from ATP. The inhibition was not competitive with Ca2+ in the case of AlFx and MgFx, in contrast with results observed in other P-ATPases. The affinities for AlFx, BeFx and MgFx increased slightly when PMCA was activated by calmodulin. On the other hand, the water content in the nucleotide binding site increased as a result of the conformational change produced by fluoride complexes binding. This effect occurred also when PMCA was phosphorylated from ATP, which allowed to measure the concentration of the phosphorylated intermediate in real time. In summary, our results show that these fluoride complexes are a useful to reveal the properties of different phosphorylated intermediates that are involved in the mechanism of hydrolysis of ATP by the PMCA.1 Gong D., Chi X., Ren K., Huang G., Zhou G., Yan N., Lei J., Zhou Q. (2018) Nat. Commun. 3623, 9 1This work was supported by Agencia Nacional de Promoción Científica y Tecnológica PICT 2014 0065, Consejo Nacional de Investigaciones Científicas y Técnicas PIP 11220150100250CO, and Universidad de Buenos Aires Ciencia y Técnica grant 2014-2017: 20020130100254B