title: Localization of the 12.6-kDa FK506-binding protein (FKBP12.6) binding site to the NH2-terminal domain of the cardiac Ca2+ release channel (ryanodine receptor).
abstract: The 12.6-kDa FK506-binding protein (FKBP12.6) interacts with the cardiac ryanodine receptor (RyR2) and modulates its channel function. However, the molecular basis of FKBP12.6-RyR2 interaction is poorly understood. To investigate the significance of the isoleucine-proline (residues 2427-2428) dipeptide epitope, which is thought to form an essential part of the FKBP12.6 binding site in RyR2, we generated single and double mutants, P2428Q, I2427E/P2428A, and P2428A/L2429E, expressed them in HEK293 cells, and assessed their ability to bind GST-FKBP12.6. None of these mutations abolished GST-FKBP12.6 binding, indicating that this isoleucine-proline motif is unlikely to form the core of the FKBP12.6 binding site in RyR2. To systematically define the molecular determinants of FKBP12.6 binding, we constructed a series of internal and NH(2)- and COOH-terminal deletion mutants of RyR2 and examined the effect of these deletions on GST-FKBP12.6 binding. These deletion analyses revealed that the first 305 NH(2)-terminal residues and COOH-terminal residues 1937-4967 are not essential for GST-FKBP12.6 binding, whereas multiple sequences within a large region between residues 305 and 1937 are required for GST-FKBP12.6 interaction. Furthermore, an NH(2)-terminal fragment containing the first 1937 residues is sufficient for GST-FKBP12.6 binding. Co-expression of overlapping NH(2) and COOH-terminal fragments covering the entire sequence of RyR2 produced functional channels but did not restore GST-FKBP12.6 binding. These data suggest that FKBP12.6 binding is likely to be conformationdependent. Binding of FKBP12.6 to the NH(2)-terminal domain may play a role in stabilizing the conformation of this region.
authors.affiliation: Cardiovascular Research Group, Department of Physiology & Biophysics, University of Calgary, Alberta T2N 4N1, Canada.
authors.forename: Haruko
authors.initials: H
authors.lastname: Masumiya
authors.forename: Ruiwu
authors.initials: R
authors.lastname: Wang
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Bailong
authors.initials: B
authors.lastname: Xiao
authors.forename: S R Wayne
authors.initials: SR
authors.lastname: Chen
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002110:Caffeine / Q000494:pharmacology; D002460:Cell Line; D017931:DNA Primers; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D011487:Protein Conformation; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D022021:Tacrolimus Binding Proteins / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D005982:Glutathione Transferase; D022021:Tacrolimus Binding Proteins; C403484:tacrolimus binding protein 1B