Sharma Manjuli Rani, Jeyakumar Loice H, Fleischer Sidney, Wagenknecht Terence
Wadsworth Center, New York State Department of Health, School of Public Health, State University of New York at Albany, Albany, New York 12201-0509, USA.
Biophys J. 2006 Jan 1;90(1):164-72. doi: 10.1529/biophysj.105.063503. Epub 2005 Oct 7.
The cardiac isoform of the ryanodine receptor (RyR2) from dog binds predominantly a 12.6-kDa isoform of the FK506-binding protein (FKBP12.6), whereas RyR2 from other species binds both FKBP12.6 and the closely related isoform FKBP12. The role played by FKBP12.6 in modulating calcium release by RyR2 is unclear at present. We have used cryoelectron microscopy and three-dimensional (3D) reconstruction techniques to determine the binding position of FKBP12.6 on the surface of canine RyR2. Buffer conditions that should favor the "open" state of RyR2 were used. Quantitative comparison of 3D reconstructions of RyR2 in the presence and absence of FKBP12.6 reveals that FKBP12.6 binds along the sides of the square-shaped cytoplasmic region of the receptor, adjacent to domain 9, which forms part of the four clamp (corner-forming) structures. The location of the FKBP12.6 binding site on "open" RyR2 appears similar, but slightly displaced (by 1-2 nm) from that found previously for FKBP12 binding to the skeletal muscle ryanodine receptor that was in the buffer that favors the "closed" state. The conformation of RyR2 containing bound FKBP12.6 differs considerably from that depleted of FKBP12.6, particularly in the transmembrane region and in the clamp structures. The x-ray structure of FKBP12.6 was docked into the region of the 3D reconstruction that is attributable to bound FKBP12.6, to show the relative orientations of amino acid residues (Gln-31, Asn-32, Phe-59) that have been implicated as being critical in interactions with RyR2. A thorough understanding of the structural basis of RyR2-FKBP12.6 interaction should aid in understanding the roles that have been proposed for FKBP12.6 in heart failure and in certain forms of sudden cardiac death.
犬源的兰尼碱受体（RyR2）的心脏亚型主要结合一种12.6 kDa的FK506结合蛋白（FKBP12.6）亚型，而其他物种的RyR2则同时结合FKBP12.6和密切相关的FKBP12亚型。目前尚不清楚FKBP12.6在调节RyR2钙释放中所起的作用。我们利用冷冻电子显微镜和三维（3D）重建技术来确定FKBP12.6在犬源RyR2表面的结合位置。采用了有利于RyR2“开放”状态的缓冲条件。对存在和不存在FKBP12.6时RyR2的3D重建进行定量比较，结果显示FKBP12.6沿着受体方形胞质区域的边缘结合，与结构域9相邻，结构域9是四个夹子（形成角的）结构的一部分。“开放”状态的RyR2上FKBP12.6结合位点的位置似乎相似，但与之前在有利于“关闭”状态的缓冲液中发现的FKBP12与骨骼肌兰尼碱受体结合的位置相比略有偏移（1 - 2 nm）。结合了FKBP12.6的RyR2的构象与去除了FKBP12.6的RyR2的构象有很大不同，特别是在跨膜区域和夹子结构中。将FKBP12.6的X射线结构对接至3D重建中可归因于结合的FKBP12.6的区域，以显示已被认为在与RyR2相互作用中起关键作用的氨基酸残基（Gln - 31、Asn - 32、Phe - 59）的相对取向。深入了解RyR2 - FKBP12.6相互作用的结构基础应有助于理解FKBP12.6在心力衰竭和某些形式的心源性猝死中所起的作用。
