title: ATP interacts with the CPVT mutation-associated central domain of the cardiac ryanodine receptor.
abstract: BACKGROUND
  This study was designed to determine whether the cardiac ryanodine receptor (RyR2) central domain, a region associated with catecholamine polymorphic ventricular tachycardia (CPVT) mutations, interacts with the RyR2 regulators, ATP and the FK506-binding protein 12.6 (FKBP12.6).
  
  
  METHODS
  Wild-type (WT) RyR2 central domain constructs (G(2236)to G(2491)) and those containing the CPVT mutations P2328S and N2386I, were expressed as recombinant proteins. Folding and stability of the proteins were examined by circular dichroism (CD) spectroscopy and guanidine hydrochloride chemical denaturation.
  
  
  RESULTS
  The far-UV CD spectra showed a soluble stably-folded protein with WT and mutant proteins exhibiting a similar secondary structure. Chemical denaturation analysis also confirmed a stable protein for both WT and mutant constructs with similar two-state unfolding. ATP and caffeine binding was measured by fluorescence spectroscopy. Both ATP and caffeine bound with an EC50 of ~200-400μM, and the affinity was the same for WT and mutant constructs. Sequence alignment with other ATP binding proteins indicated the RyR2 central domain contains the signature of an ATP binding pocket. Interaction of the central domain with FKBP12.6 was tested by glutaraldehyde cross-linking and no association was found.
  
  
  CONCLUSIONS
  The RyR2 central domain, expressed as a 'correctly' folded recombinant protein, bound ATP in accord with bioinformatics evidence of conserved ATP binding sequence motifs. An interaction with FKBP12.6 was not evident. CPVT mutations did not disrupt the secondary structure nor binding to ATP.
  
  
  CONCLUSIONS
  Part of the RyR2 central domain CPVT mutation cluster, can be expressed independently with retention of ATP binding.
authors.affiliation: Institute of Molecular and Experimental Medicine, Cardiff University, Cardiff, UK. blayney@cf.ac.uk
authors.forename: Lynda
authors.initials: L
authors.lastname: Blayney
authors.forename: Konrad
authors.initials: K
authors.lastname: Beck
authors.forename: Ewan
authors.initials: E
authors.lastname: MacDonald
authors.forename: Leon
authors.initials: L
authors.lastname: D'Cruz
authors.forename: Michail
authors.initials: M
authors.lastname: Nomikos
authors.forename: Julia
authors.initials: J
authors.lastname: Griffiths
authors.forename: Angelos
authors.initials: A
authors.lastname: Thanassoulas
authors.forename: George
authors.initials: G
authors.lastname: Nounesis
authors.forename: F Anthony
authors.initials: FA
authors.lastname: Lai
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D002942:Circular Dichroism; D006801:Humans; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D022021:Tacrolimus Binding Proteins / Q000378:metabolism
chemical_list: D019837:Ryanodine Receptor Calcium Release Channel; D000255:Adenosine Triphosphate; D022021:Tacrolimus Binding Proteins; C403484:tacrolimus binding protein 1B
keywords: ATP binding motifs; ATP binding site; CPVT; Caffeine; Cardiac ryanodine receptor; Catecholaminergic polymorphic ventricular tachycardia (CPVT) mutations; FKBP12.6; PDB; RCD; WT; catecholaminergic polymorphic ventricular tachycardia; protein data bank; recombinant central domain; wild type