The structural properties of the phosphorylated state in the Plasma Membrane Calcium Pump

DE SAUTU, MARILINA; ROSSI JUAN PABLO F.C.; SAFFIOTI NICOLÁS; BERLIN, JOSHUA; MANGIALAVORI IRENE; ROSSI ROLANDO; FERREIRA GOMES, MARIELA

SANTOS

Congreso; XLII Congresso da Sociedade Brasileira de Biofísica; 2017

Sociedad Brasilera de Biofísica

Plasma Membrane Calcium Pump (PMCA) belongs to the P-ATPase family and is a key participant in calcium homeostasis. Although some of their isoforms are essential for many biological processes and have been related to many diseases1 the structure and mechanism of function of this protein has not been fully elucidated yet. As PMCA undergoes many structural changes in its reaction cycle, we wish to stabilize the pump in different reaction cycle intermediates and study their structure. For this porpoise, we employed fluoride complexes of beryllium, aluminum and magnesium, which stabilize different analogues of phosphorylated state in P-ATPases. Fluoride complexes have been widely used to study members of this family, like the sarcoplasmic reticulum calcium pump (SERCA), but never in the PMCA. In this work, we characterize the mechanism by which fluoride complexes inhibit PMCA activity where they behave as slow reversible inhibitors. We propose a kinetic model in order to explain the inhibitory effect of each complex. We also study the nucleotide biding region of PMCA inhibited by fluoride complexes using different fluorescent probes. Our results show that fluorescent probes in the nucleotide biding domain have less quantum yield. This corresponds to a more exposed region to aqueous solvent when the protein is in its phosphorylated analogue conformations. The change in this region depends on the fluoride complex used, being maximal in presence of beryllium fluoride, which is proposed to stabilize P-ATPases with phosphate covalently bound. The conformational change associated with an intermediary of the reaction cycle also allows us to develop a method measure PMCA activity by a fluorescence approach. Finally, we run molecular dynamics simulations of the cytoplasmic domains of PMCA modelled by homology on other P-ATPases to understand the changes in the nucleotide-binding domain during the catalytic cycle.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: 20020130100254B1Brini, M., Calì, T., Ottolini, D. and Carafoli, E. (2013), The plasma membrane calcium pump in health and disease. FEBS J, 280: 5385?5397. doi:10.1111/febs.12193