title: Mapping the ryanodine receptor FK506-binding protein subunit using fluorescence resonance energy transfer.
abstract: The 12-kDa FK506-binding proteins (FKBP12 and FKBP12.6) are regulatory subunits of ryanodine receptor (RyR) Ca(2+) release channels. To investigate the structural basis of FKBP interactions with the RyR1 and RyR2 isoforms, we used site-directed fluorescent labeling of FKBP12.6, ligand binding measurements, and fluorescence resonance energy transfer (FRET). Single-cysteine substitutions were introduced at five positions distributed over the surface of FKBP12.6. Fluorescent labeling at position 14, 32, 49, or 85 did not affect high affinity binding to the RyR1. By comparison, fluorescent labeling at position 41 reduced the affinity of FKBP12.6 binding by 10-fold. Each of the five fluorescent FKBPs retained the ability to inhibit [(3)H]ryanodine binding to the RyR1, although the maximal extent of inhibition was reduced by half when the label was attached at position 32. The orientation of FKBP12.6 bound to the RyR1 and RyR2 was examined by measuring FRET from the different labeling positions on FKBP12.6 to an acceptor attached within the RyR calmodulin subunit. FRET was dependent on the position of fluorophore attachment on FKBP12.6; however, for any given position, the distance separating donors and acceptors bound to RyR1 versus RyR2 did not differ significantly. Our results show that FKBP12.6 binds to RyR1 and RyR2 in the same orientation and suggest new insights into the discrete structural domains responsible for channel binding and inhibition. FRET mapping of RyR-bound FKBP12.6 is consistent with the predictions of a previous cryoelectron microscopy study and strongly supports the proposed structural model.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Razvan L
authors.initials: RL
authors.lastname: Cornea
authors.forename: Florentin R
authors.initials: FR
authors.lastname: Nitu
authors.forename: Montserrat
authors.initials: M
authors.lastname: Samsó
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Fruen
mesh_terms: D000818:Animals; D002147:Calmodulin / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D020104:Immunophilins / Q000737:chemistry; D018482:Muscle, Skeletal / Q000378:metabolism; D009154:Mutation; D009206:Myocardium / Q000473:pathology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012433:Ryanodine / Q000737:chemistry; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013552:Swine; D022021:Tacrolimus Binding Proteins / Q000737:chemistry*
chemical_list: D002147:Calmodulin; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D022021:Tacrolimus Binding Proteins; D020104:Immunophilins
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