E2P-like states of plasma membrane Ca‑ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues.

Author(s) Saffioti, N.A.; de Sautu, M.; Ferreira-Gomes, M.S.; Rossi, R.C.; Berlin, J.; Rossi, J.Pablo F.C.; Mangialavori, I.C.
Journal Biochim Biophys Acta Biomembr
Date Published 2019 Feb 01
Abstract

The plasma membrane Ca‑ATPase (PMCA) belongs to the family of P-type ATPases, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. The crystal structure of PMCA is currently lacking. Its abundance is approximately 0.1% of the total protein in the membrane, hampering efforts to produce suitable crystals for X-ray structure analysis. In this work we characterized the effect of beryllium fluoride (BeF), aluminium fluoride (AlF) and magnesium fluoride (MgF) on PMCA. These compounds are known inhibitors of P-type ATPases that stabilize E2P ground, E2·P phosphoryl transition and E2·P product states. Our results show that the phosphate analogues BeF, AlF and MgF inhibit PMCA Ca‑ATPase activity, phosphatase activity and phosphorylation with high apparent affinity. Ca‑ATPase inhibition by AlF and BeF depended on Mg concentration indicating that this ion stabilizes the complex between these inhibitors and the enzyme. Low pH increases AlF and BeF but not MgF apparent affinity. Eosin fluorescent probe binds with high affinity to the nucleotide binding site of PMCA. The fluorescence of eosin decreases when fluoride complexes bind to PMCA indicating that the environment of the nucleotide binding site is less hydrophobic in E2P-like states. Finally, measuring the time course of E → E2P-like conformational change, we proposed a kinetic model for the binding of fluoride complexes and vanadate to PMCA. In summary, our results show that these fluoride complexes reveal different states of phosphorylated intermediates belonging to the mechanism of hydrolysis of ATP by the PMCA.

DOI 10.1016/j.bbamem.2018.11.001
ISSN 1879-2642
Citation Saffioti NA, de Sautu M, Ferreira-Gomes MS, Rossi RC, Berlin J, Rossi JP, et al. E2P-like states of plasma membrane Ca‑ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues. Biochim Biophys Acta Biomembr. 2019;1861(2):366-379.

Related Applications, Forms & Industries