Lee Eun Hui, Rho Seong-Hwan, Kwon Soon-Jae, Eom Soo Hyun, Allen Paul D, Kim Do Han
Department of Life Science, Kwangju Institute of Science and Technology, Gwangju 500-712 Korea.
J Biol Chem. 2004 Jun 18;279(25):26481-8. doi: 10.1074/jbc.M309574200. Epub 2004 Mar 19.
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.
已知两种FK506结合蛋白FKBP12和FKBP12.6分别与骨骼肌的兰尼碱受体（RyR1）和心肌的兰尼碱受体（RyR2）紧密相关，它们之间的相互作用对RyR的通道功能很重要。就心肌而言，FKBP12.6的三个氨基酸残基（Gln-31、Asn-32和Phe-59）对于与RyR2的选择性结合可能至关重要（辛，H.B.，罗杰斯，K.，齐，Y.，金松，T.，和弗莱舍尔，S.（1999年）《生物化学杂志》274卷，15315 - 15319页）。在本研究中，为了确定FKBP12中对与RyR1选择性结合重要的氨基酸残基，我们对FKBP12中与FKBP12.6对应残基不同的9个氨基酸残基进行了突变（Q3E、R18A、E31Q、D32N、M49R、R57A、W59F、H94A和K105A），并通过使用突变体的谷胱甘肽S - 转移酶融合蛋白和经Triton X - 100增溶、去除FKBP12的兔骨骼肌肌质网囊泡进行体外结合测定，来检测这些突变体与RyR1的结合特性。在所测试的9个突变体中，只有Q3E和R18A失去了它们与RyR1的选择性结合能力。此外，通过对由引入不同大小、电荷和疏水性产生的9个位置的33种不同突变体与RyR1进行共免疫沉淀发现，由不可替代的Gln - 3形成的氢键整合以及由Arg - 18和Met - 49残基形成的疏水相互作用可能是FKBP12与RyR1结合的一种机制。因此，这些结果表明，FKBP12的N端区域（Gln - 3和Arg - 18）以及Met - 49对于FKBP12在骨骼肌中与RyR1的结合至关重要且具有独特性。
