E2P-like states of Plasma Membrane Ca2+-ATPase CHARACTERIZATION OF VANADATE AND FLUORIDE-STABILIZED PHOSPHOENZYME ANALOGUES

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

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2019 vol. 1861 p. 366 - 379

0005-2736

The plasma membrane Ca2+-ATPase (PMCA) belongs to the family ofP-type ATPases, which share the formation of an acid-stablephosphorylated intermediate as part of their reaction cycle. The crystalstructure of PMCA is currently lacking. Its abundance is approximately0.1% of the total protein in the membrane, hampering efforts to producesuitable crystals for X-ray structure analysis. In this work wecharacterized the effect of beryllium fluoride (BeFx), aluminum fluoride(AlFx) and magnesium fluoride (MgFx) on PMCA. These compounds are knowninhibitors of P-type ATPases that stabilize E2P ground, E2∙P phosphoryltransition and E2∙Pi product states. Our results show that the phosphateanalogues BeFx, AlFx and MgFx inhibit PMCA Ca2+-ATPase activity,phosphatase activity and phosphorylation with high apparent affinity.Ca2+-ATPase inhibition by AlFx and BeFx depended on Mg2+ concentrationindicating that this ion stabilizes the complex between these inhibitorsand the enzyme. Low pH increases AlFx and BeFx but not MgFx apparentaffinity. Eosin fluorescent probe binds with high affinity to thenucleotide binding site of PMCA. The fluorescence of eosin decreases whenfluoride complexes bind to PMCA indicating that the environment of thenucleotide binding site is less hydrophobic in E2P-like states. Finally,measuring the time course of E→E2P-like conformational change, weproposed a kinetic model for the binding of fluoride complexes andvanadate to PMCA.In summary, our results show that these fluoride complexes revealdifferent states of phosphorylated intermediates belonging to themechanism of hydrolysis of ATP by the PMCA