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)

• Published in: The Journal of biological chemistry Vol. 278; no. 6; pp. 3786 - 3792
• Main Authors: Masumiya, Haruko, Wang, Ruiwu, Zhang, Jing, Xiao, Bailong, Chen, S. R. Wayne
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07.02.2003; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Base Sequence; Binding Sites; Caffeine - pharmacology; Cell Line; DNA Primers; Glutathione Transferase - metabolism; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Myocardium - metabolism; Protein Conformation; Ryanodine Receptor Calcium Release Channel - chemistry; Ryanodine Receptor Calcium Release Channel - drug effects; Ryanodine Receptor Calcium Release Channel - metabolism; Sequence Homology, Amino Acid; Tacrolimus Binding Proteins - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M210962200

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 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 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 conformationdependent. Binding of FKBP12.6 to the NH2-terminal domain may play a role in stabilizing the conformation of this region.