N-terminal Region of FKBP12 Is Essential for Binding to the Skeletal Ryanodine Receptor

Abstract

It is known that the two types of FK506-binding proteins FKBP12 and FKBP12.6 are tightly associated with the skeletal (RyR1) and cardiac ryanodine receptors (RyR2), respectively, and their interactions are important for channel functions of the RyR. In the case of cardiac muscle, three amino acid residues (Gln-31, Asn-32, and Phe-59) of FKBP12.6 could be essential for the selective binding to RyR2 (Xin, H. B., Rogers, K., Qi, Y., Kanematsu, T., and Fleischer, S. (1999) J. Biol. Chem. 274, 15315-15319). In this study to identify amino acid residues of FKBP12 that are important for the selective binding to RyR1, we mutated 9 amino acid residues of FKBP12 that differ from the counterparts of FKBP12.6 (Q3E, R18A, E31Q, D32N, M49R, R57A, W59F, H94A, and K105A), and we examined binding properties of these mutants to RyR1 by in vitro binding assay by using glutathione S-transferase-fused proteins of the mutants and Triton X-100-solubilized, FKBP12-depleted rabbit skeletal sarcoplasmic reticulum vesicles. Among the nine mutants tested, only Q3E and R18A lost their selective binding ability to RyR1. Furthermore, co-immunoprecipitation of RyR1 with 33 various mutants for the 9 positions produced by introducing different size, charge, and hydrophobicity revealed that an integration of the hydrogen bonds by the irreplaceable Gln-3 and the hydrophobic interactions by the residues Arg-18 and Met-49 could be a possible mechanism for the binding of FKBP12 to RyR1. Therefore, these results suggest that the N-terminal regions of FKBP12 (Gln-3 and Arg-18) and Met-49 are essential and unique for binding of FKBP12 to RyR1 in skeletal muscle.

Analysis of calstabin2 (FKBP12.6)–ryanodine receptor interactions: Rescue of heart failure by calstabin2 in mice

“…Residues outside of the calstabin2 binding pocket likely also make contacts with RyR2. Indeed, calstabin2-Q3, calstabin2-R18, and calstabin2-M49 have also been proposed to play a role in bind to RyR2 (22). The existence of multiple interaction sites between calstabin and RyR is likely the reason that binding studies with fragments of RyR have been inconsistent (23,24).…”

“…To evaluate the ability of calstabin2 mutants to bind to PKAphosphorylated RyR2, RyR2 from CSR membranes were maximally phosphorylated by using exogenous PKA (12). As a negative control, CSR membranes were treated with PKA in the presence of a specific PKA inhibitor (PKI [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] ) (data not shown). Calstabin2-D37S and calstabin2-D37V retained the ability to bind to PKA-phosphorylated RyR2 (Fig.…”

Differential Responses of the Backbone and Side-Chain Conformational Dynamics in FKBP12 upon Binding the Transition-State Analog FK506: Implications for Transition-State Stabilization and Target Protein Recognition

“…† www.pdb.org methylene groups forming a hydrophobic surface patch that has been implicated in the binding of RyR1. 67 It seems likely that residues Q3, R18, and M49 are involved in interactions that govern the selective binding of RyR1 over RyR2. However, a low-resolution (16 Å) cryo-electron microscopy model of the FKBP12-RyR1 complex suggests that Q3, E31, and D32 of FKBP12 face RyR1, while R18 and M49 point away from the modeled interface.…”

Ablation of Skeletal Muscle Triadin Impairs FKBP12/RyR1 Channel Interactions Essential for Maintaining Resting Cytoplasmic Ca2+

“…Immunoprecipitation-Co-immunoprecipitation assays were performed according to the methods of Lee et al (24,25). Briefly, 300 g of crude membrane homogenate were solubilized 45 min in 10 mM Tris/HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 mM Na 3 VO 4 , 1% Triton X-100, and 1% digitonin, supplemented with protease inhibitors.…”