Structure of different phosphorylated states of Plasma Membrane Calcium Pump

NICOLAS ANDRÉS SAFFIOTI; MARILINA DE SAUTU; ROLANDO ROSSI; JOSUA BERLIN; JUAN PABLO FC ROSSI; IRENE MANGIALAVORI

Congreso; Reunión conjunta de las Sociedades de Biociencias; 2017

The control of cytoplasmic calcium concentration relies on many components. One of the most important is the Plasma Membrane Calcium Pump (PMCA) which belongs to the P-ATPase family. Structure and mechanism of function of this protein has not been fully elucidated yet, because X-ray crystal diffraction and NMR technique have not been successful.To elucidate which structural changes are produced in PMCA reaction cycle we stabilized the pump in different reaction cycle stages and studied their structure. For this, we employed fluoride complexes of beryllium, aluminum and magnesium, each one stabilizes different analogues of phosphorylated intermediary in P-ATPases but they have never been assayed on PMCA.By fluorescence experiments using eosin which binds to the N domain of PMCA, we studied the structural changes upon fluoride complex binding. Quantum yield of the probe bound to the protein decreased in the complexes presence. This change depended on the presence of calcium and the complex identity. This indicates that different analogues of the phosphorylated state in PMCA are achievable with this strategy.Determining the kinetics of the conformational change, we propose a model of how these complexes bind and stabilize phosphorylated states in PMCA. The detectable conformational change associated with an intermediary of the reaction cycle also allows us to develop a method to measure PMCA activity by a fluorescence approach. Finally, we prepared and refined PMCA structural models based on homology with Sarcoplasmic Reticulum Calcium Pump (SERCA) in different states. This approach helped us to understand the changes in the nucleotide-binding domain during the catalytic cycle of PMCA.