E2P-like states of plasma membrane Ca2+‑ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues

Publication date: Available online 9 November 2018Source: Biochimica et Biophysica Acta (BBA) - BiomembranesAuthor(s): Nicolás A. Saffioti, Marilina de Sautu, Mariela S. Ferreira-Gomes, Rolando C. Rossi, Joshua Berlin, Juan Pablo F.C. Rossi, Irene C. MangialavoriAbstractThe plasma membrane Ca2+‑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 (BeFx), aluminium fluoride (AlFx) and magnesium fluoride (MgFx) on PMCA. These compounds are known inhibitors of P-type ATPases that stabilize E2P ground, E2·P phosphoryl transition and E2·Pi product states. Our results show that the phosphate analogues 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+ concentration indicating that this ion stabilizes the complex between these inhibitors and the enzyme. Low pH increases AlFx and BeFx but not MgFx 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 indic...
Source: Biochimica et Biophysica Acta (BBA) Biomembranes - Category: Biochemistry Source Type: research