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

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Abstract

The 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 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.

Original languageEnglish (US)
Pages (from-to)366-379
Number of pages14
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1861
Issue number2
DOIs
StatePublished - Feb 1 2019

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Vanadates
Calcium-Transporting ATPases
Cell membranes
Fluorides
Cell Membrane
Eosine Yellowish-(YS)
Adenosine Triphosphatases
Nucleotides
Binding Sites
Phosphorylation
Enzyme Inhibitors
Fluorescent Dyes
Phosphoric Monoester Hydrolases
Hydrolysis
Membrane Proteins
Adenosine Triphosphate
Crystal structure
Fluorescence
Phosphates
X-Rays

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Cell Biology

Keywords

  • Metal fluorides
  • PMCA
  • Phosphorylated state
  • P‑ATPases
  • Reaction cycle

Cite this

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title = "E2P-like states of plasma membrane Ca2+‑ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues",
abstract = "The 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 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.",
keywords = "Metal fluorides, PMCA, Phosphorylated state, P‑ATPases, Reaction cycle",
author = "Joshua Berlin",
year = "2019",
month = "2",
day = "1",
doi = "https://doi.org/10.1016/j.bbamem.2018.11.001",
language = "English (US)",
volume = "1861",
pages = "366--379",
journal = "Biochimica et Biophysica Acta - Biomembranes",
issn = "0005-2736",
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number = "2",

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TY - JOUR

T1 - E2P-like states of plasma membrane Ca2+‑ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues

AU - Berlin, Joshua

PY - 2019/2/1

Y1 - 2019/2/1

N2 - The 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 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.

AB - The 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 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.

KW - Metal fluorides

KW - PMCA

KW - Phosphorylated state

KW - P‑ATPases

KW - Reaction cycle

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SN - 0005-2736

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