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)

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 NH2- 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 NH2-terminal residues and COOH-terminal residues 19374967 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 NH2-terminal fragment containing the first 1937 residues is sufficient for GST-FKBP12.6 binding. Co-expression of overlapping NH2 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 conformation-dependent. Binding of FKBP12.6 to the NH2-terminal domain may play a role in stabilizing the conformation of this region.