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

NICOLAS ANDRÉS SAFFIOTI; MARILINA DE SAUTU; ROLANDO ROSSI; JOSUA BERLIN; JUAN PABLO FC ROSSI; MARIELA FERREIRA-GOMES; IRENE CECILIA MANGIALAVORI

Congreso; XLII Congreso da Sociedade Brasileira de Biofisica; 2017

The structural properties of the phosphorylated state in the Plasma Membrane Calcium PumpNicolas Andres Saffioti1, Marilina de Sautu1, Rolando C. Rossi1, Joshua R. Berlin2,Juan Pablo F. Rossi1, Mariela Ferreira Gomes1, Irene C. Mangialavori1.1 Departamento de Química Biológica, IQUIFIB, Capital Federal, Buenos Aires, Argentina, 2 Department of Pharmacology and Physiology, New Jersey. Medical School, Rutgers University, Newark, NJ, USA.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 for this inhibition for each complex. We also studied the nucleotide biding region of PMCA in its phosphorylated analogue conformation 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 associates with an intermediary of the reaction cycle also allow us to develop a method measure PMCA activity by a fluorescence approach. Finally, we ran 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.With grants from ANPCYT, CONICET and UBACYT1Brini, 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