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Critical roles of interdomain interactions for modulatory ATP binding to sarcoplasmic reticulum Ca2+-ATPase

Johannes D Clausen1, Anne Nyholm Holdensen1, Jens Peter Andersen2

  • 1From the Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark, and the Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, Department of Molecular Biology and Genetics, Aarhus University, DK-8000 Aarhus C, Denmark.

The Journal of Biological Chemistry
|September 7, 2014
PubMed

Insights

Adenosine triphosphate (ATP) modulates sarcoplasmic reticulum Ca(2+)-ATPase dephosphorylation by stabilizing the transition state. Specific interdomain interactions, like Asp(203)-Arg(678), are crucial for this ATP-stimulated dephosphorylation mechanism.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) utilizes adenosine triphosphate (ATP) for calcium ion (Ca(2+)) transport.
  • ATP has dual roles in the SERCA reaction cycle: phosphorylation and non-phosphorylating modulation.
  • The modulatory role of ATP enhances dephosphorylation and overall Ca(2+) transport efficiency.

Purpose of the Study:

  • To elucidate the molecular mechanism by which ATP modulates the dephosphorylation step in Ca(2+)-ATPase.
  • To identify specific amino acid residues and interdomain interactions critical for ATP-stimulated dephosphorylation.

Main Methods:

  • Site-directed mutagenesis of key residues (Ser(186), Asp(203), Glu(439), Arg(678)) in Ca(2+)-ATPase.
  • Construction and characterization of wild-type and mutant Ca(2+)-ATPases.
  • Formation of stable complexes with aluminum fluoride (E2·AlF) and beryllium fluoride (E2·BeF) to mimic dephosphorylation intermediates.
  • Assessment of ATP binding affinities to these intermediates.

Main Results:

  • Single mutations of Ser(186) or Asp(203) abolished ATP stimulation of dephosphorylation.
  • The Asp(203)-Arg(678) interdomain interaction is critical for ATP stimulation, while Ser(186)-Glu(439) is not sufficient.
  • ATP binding affinity is higher for the E2·P transition state than the E2P ground state in wild-type enzyme, indicating transition state stabilization.
  • The Asp(203)-Arg(678) and Ser(186)-Glu(439) bonds stabilize ATP interaction in the E2·P state; Ser(186)-Glu(439) binding is mutually exclusive with ATP in the E2P state.

Conclusions:

  • The modulatory effect of ATP on Ca(2+)-ATPase dephosphorylation is mediated by nucleotide-induced transition state stabilization.
  • Specific interdomain interactions, particularly Asp(203)-Arg(678), are essential for tightening ATP interaction in the transition state.
  • The interplay between ATP binding and interdomain bonds dictates the enzyme's conformational states and catalytic cycle efficiency.
Keywords:
ATPaseCalcium ATPaseEnzyme KineticsMembrane EnergeticsMembrane EnzymeMembrane TransportPhosphorylationSite-directed Mutagenesis

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