4.0-Å resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement
4.0-Å resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT reveals its unique subunit arrangement
复制标题
DOI：
10.1073/pnas.0913774107
复制
发表时间：
2010-03-16
影响因子：
11.1
通讯作者：
Chiu, Wah
中科院分区：
文献类型：
--
作者：
Cong, Yao;Baker, Matthew L.;Chiu, Wah
真核生物必需的双环伴侣蛋白TRiC/CCT（TCP1环复合物或含TCP1的伴侣蛋白）协助约5 - 10%的细胞蛋白质组折叠。许多TRiC底物无法由原核生物或古细菌的其他伴侣蛋白折叠。这些独特的折叠特性可能与TRiC独特的异源寡聚亚基组织有关，其中每个环由八个不同的同源亚基组成，其排列方式仍不确定。我们在不施加对称性的情况下使用单颗粒冷冻电镜，以4.7埃的分辨率确定了哺乳动物TRiC的结构。这揭示了其两个环之间存在一个2重轴，导致环之间有两种同型亚基相互作用。随后的一个2重对称图谱产生了一个4.0埃分辨率的结构，该结构显示了大部分侧链、环和插入片段的密度。尽管这些亚基序列相似，但这些特征使我们能够明确识别所有八个单个亚基。独立的生化近邻分析支持我们从冷冻电镜得出的TRiC亚基排列。我们从一个初始的同源模型出发，根据冷冻电镜密度进行优化，为每个亚基获得了一个Cα主链模型。随后一个亚基的优化原子模型显示，在拉氏图的允许区域内，约95%的主链二面角符合要求。TRiC亚基排列的确定为理解其独特功能和机制开辟了道路。特别是，TRiC封闭折叠腔内壁不均匀分布的带正电荷的壁与原核生物和古细菌的伴侣蛋白显著不同。这些内表面化学性质可能在TRiC的细胞底物特异性中起重要作用。
The essential double-ring eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) assists the folding of similar to 5-10% of the cellular proteome. Many TRiC substrates cannot be folded by other chaperonins from prokaryotes or archaea. These unique folding properties are likely linked to TRiC's unique hetero-oligomeric subunit organization, whereby each ring consists of eight different paralogous subunits in an arrangement that remains uncertain. Using single particle cryo-EM without imposing symmetry, we determined the mammalian TRiC structure at 4.7 angstrom resolution. This revealed the existence of a 2-fold axis between its two rings resulting in two homotypic subunit interactions across the rings. A subsequent 2-fold symmetrized map yielded a 4.0-angstrom resolution structure that evinces the densities of a large fraction of side chains, loops, and insertions. These features permitted unambiguous identification of all eight individual subunits, despite their sequence similarity. Independent biochemical near-neighbor analysis supports our cryo-EM derived TRiC subunit arrangement. We obtained a C alpha backbone model for each subunit from an initial homology model refined against the cryo-EM density. A subsequently optimized atomic model for a subunit showed similar to 95% of the main chain dihedral angles in the allowable regions of the Ramachandran plot. The determination of the TRiC subunit arrangement opens the way to understand its unique function and mechanism. In particular, an unevenly distributed positively charged wall lining the closed folding chamber of TRiC differs strikingly from that of prokaryotic and archaeal chaperonins. These interior surface chemical properties likely play an important role in TRiC's cellular substrate specificity.