Structure of different phosphorylated states of Plasma Membrane Calcium Pump

SAFFIOTI, N. A.; DE SAUTU, M.; ROSSI, R.C.; BERLIN, J. R.; ROSSI, J. P. F. C.; FERREIRA-GOMES, M. S.; MANGIALAVORI, I. C.

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

The control of the cytoplasmic calcium homeostasis relies on many transporters like the Plasma Membrane Calcium Pump (PMCA) and the Sarcoplasmic Reticulum Calcium Pump (SERCA) which belong to the P-ATPase family. At difference with SERCA, structure and function of PMCA has not been fully elucidated yet, because obtaining suitable preparations for X-ray crystal diffraction and NMR techniques was unsuccessful. To elucidate the structural changes produced in PMCA during the reaction cycle we tested a purified preparation of the pump with fluoride complexes of beryllium, aluminum and magnesium, each one stabilizing different analogues of phosphorylated intermediates in P-ATPases. These blockers were previously assayed in SERCA leading to different conformational states. To follow the binding of fluoride complexes by fluorescence we employed eosin, which binds to the N domain of PMCA. Quantum yield of the bound probe decreased in the presence of fluoride complexes indicating more exposure to solvent. The magnitude of this change depended on the presence of calcium and the complex identity. Following the kinetics of the conformational change, we propose a model to explain how these complexes stabilize phosphorylated states in PMCA. The detectable conformational change associated with an intermediate of the reaction cycle also allows us to develop a method to measure PMCA activity using a fluorescence approach. Finally, we prepared and refined PMCA structural models based on homology with SERCA and Na+/K+ ATPase at different states, to understand the changes at the nucleotide-binding domain during the catalytic cycle of PMCA.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