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ATP interacts with the CPVT mutation-associated central domain of the cardiac ryanodine receptor

Lynda Blayney1, Konrad Beck, Ewan MacDonald

  • 1Institute of Molecular and Experimental Medicine, Cardiff University, Cardiff, UK. blayney@cf.ac.uk

Biochimica Et Biophysica Acta
|June 11, 2013
PubMed
Abstract

Insights

The cardiac ryanodine receptor central domain binds ATP, and CPVT mutations do not affect this interaction or protein structure. FKBP12.6 does not associate with this RyR2 domain.

Area of Science:

  • Cardiovascular Biology
  • Molecular Cardiology
  • Protein Structure and Function

Background:

  • The cardiac ryanodine receptor (RyR2) central domain is implicated in catecholamine polymorphic ventricular tachycardia (CPVT).
  • Understanding RyR2 interactions with ATP and FKBP12.6 is crucial for CPVT pathogenesis.

Purpose of the Study:

  • To investigate the interaction of the RyR2 central domain with ATP and FKBP12.6.
  • To determine the effect of CPVT mutations on RyR2 central domain structure and function.

Main Methods:

  • Recombinant expression of wild-type and CPVT mutant RyR2 central domain constructs.
  • Circular dichroism spectroscopy and chemical denaturation for protein folding and stability assessment.
  • Fluorescence spectroscopy for ATP and caffeine binding assays; glutaraldehyde cross-linking for FKBP12.6 interaction studies.

Main Results:

  • Wild-type and CPVT mutant RyR2 central domains exhibited stable, correctly folded structures with similar secondary structures.
  • ATP and caffeine binding occurred with similar affinities (~200-400μM EC50) for both wild-type and mutant constructs.
  • Bioinformatics analysis suggested an ATP binding pocket within the RyR2 central domain; no interaction with FKBP12.6 was detected.

Conclusions:

  • The RyR2 central domain, when expressed as a recombinant protein, retains ATP binding capacity, supported by sequence motifs.
  • CPVT mutations do not compromise the secondary structure or ATP binding of the RyR2 central domain.
  • The RyR2 central domain does not appear to interact with FKBP12.6.
Keywords:
ATP binding motifsATP binding siteCPVTCaffeineCardiac ryanodine receptorCatecholaminergic polymorphic ventricular tachycardia (CPVT) mutationsFKBP12.6PDBRCDWTcatecholaminergic polymorphic ventricular tachycardiaprotein data bankrecombinant central domainwild type

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