Conformational selective binding of metal fluoride complexes to the Plasma Membrane Ca2+-ATPase and structure stabilization

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

Congreso; XLVIII Reunión Annual de la Sociedad de Biofísica Argentina.; 2019

The Plasma Membrane Ca2+ ATPase (PMCA) is a key participant in cytoplasmic Ca2+ regulation. As others P-ATPases, PMCA transports Ca2+ by the Albers and Post mechanism. In this, proteins have two main conformations, E1 and E2, and phosphorylate from ATP to transport ions against their electrochemical gradient. Recently, we demonstrated that fluoride complexes of aluminium (AlFx), beryllium (BeFx) and magnesium (MgFx) stabilize conformations very similar to the phosphorylated intermediate of PMCA1. The aim of this work is to understand what conformational changes occur upon the phosphorylation of PMCA. To this end, we measure the fluorescence of the probe eosin, which binds with high affinity to the nucleotide binding site of PMCA. When the protein binds fluoride complexes, eosin fluorescence decreases, and the same effect is seen when the pump phosphorylates from ATP in presence of Ca2+. However, during transport activity, part of the fluorescence decrease is due to displacement of eosin by ADP from its binding site. Our results show that the ADP and ATP affinity for PMCA in E2 conformation are 172 ± 28 and 74 ± 17 µM, respectively. The binding of fluoride complexes to PMCA depended strictly on Mg2+ concentration. Furthermore, these fluoride complexes only binds to PMCA-Mg2+. This property allowed us to measure the PMCA affinity for Mg2+ (2,2 ± 0,4 mM) in E2 conformation. Finally, we studied thermal stability of PMCA by measuring the enzyme activity as a function of time. Fluoride complexes stabilize PMCA structure and delay activity loss when the protein is incubated at 44°C compared to the free protein. We can conclude that fluoride complexes are ligands of PMCA-Mg2+ complex, they set PMCA structure in a conformation analogous to E2-P, stabilizing it. This property allows to study the PMCA structure in E2 conformations.1 Saffioti N.A, de Sautu M., Ferreira-Gomes M.S., Rossi R.C., Berlin J., Rossi J.P., Mangialavori, I.C. (2019) BBA-Biomembranes. 1861, 366-379.This 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