Department of Structural Biology, Stanford University School of Medicine, D100 Fairchild Building, Stanford, CA 94305, USA.
Proc Natl Acad Sci U S A. 2012 Feb 21;109(8):2884-9. doi: 10.1073/pnas.1119472109. Epub 2012 Feb 1.
The TRiC/CCT chaperonin is a 1-MDa hetero-oligomer of 16 subunits that assists the folding of proteins in eukaryotes. Low-resolution structural studies confirmed the TRiC particle to be composed of two stacked octameric rings enclosing a folding cavity. The exact arrangement of the different proteins in the rings underlies the functionality of TRiC and is likely to be conserved across all eukaryotes. Yet despite its importance it has not been determined conclusively, mainly because the different subunits appear nearly identical under low resolution. This work successfully addresses the arrangement problem by the emerging technique of cross-linking, mass spectrometry, and modeling. We cross-linked TRiC under native conditions with a cross-linker that is primarily reactive toward exposed lysine side chains that are spatially close in the context of the particle. Following digestion and mass spectrometry we were able to identify over 60 lysine pairs that underwent cross-linking, thus providing distance restraints between specific residues in the complex. Independently of the cross-link set, we constructed 40,320 (= 8 factorial) computational models of the TRiC particle, which exhaustively enumerate all the possible arrangements of the different subunits. When we assessed the compatibility of each model with the cross-link set, we discovered that one specific model is significantly more compatible than any other model. Furthermore, bootstrapping analysis confirmed that this model is 10 times more likely to result from this cross-link set than the next best-fitting model. Our subunit arrangement is very different than any of the previously reported models and changes the context of existing and future findings on TRiC.
TRiC/CCT 分子伴侣是由 16 个亚基组成的 1MDa 异源寡聚体，有助于真核生物中蛋白质的折叠。低分辨率结构研究证实，TRiC 颗粒由两个堆叠的八聚体环组成，环内包含一个折叠腔。环中不同蛋白质的确切排列是 TRiC 功能的基础，并且可能在所有真核生物中都保守。尽管它很重要，但由于不同的亚基在低分辨率下几乎相同，因此尚未得出明确的结论。这项工作通过新兴的交联、质谱和建模技术成功地解决了排列问题。我们在天然条件下用交联剂交联 TRiC，交联剂主要与颗粒中空间上接近的暴露的赖氨酸侧链反应。在消化和质谱分析之后，我们能够鉴定出 60 多个赖氨酸对发生了交联，从而为复合物中特定残基之间提供了距离约束。独立于交联组，我们构建了 40320（=8 阶乘）个 TRiC 颗粒的计算模型，详尽地列举了不同亚基的所有可能排列。当我们评估每个模型与交联组的兼容性时，我们发现一个特定的模型比任何其他模型都更兼容。此外，引导分析证实，与下一个最佳拟合模型相比，该模型从交联组得出的可能性高 10 倍。我们的亚基排列与以前报道的任何模型都非常不同，改变了现有和未来关于 TRiC 的发现的背景。
