Cilia are ubiquitous eukaryotic organelles impotant for cellular motility, signaling, and sensory reception. Cilium formation requires intraflagellar transport of structural and signaling components and involves 22 different proteins organized into intraflagellar transport (IFT) complexes IFT-A and IFT-B that are transported by molecular motors. The IFT-B complex constitutes the backbone of polymeric IFT trains carrying cargo between the cilium and the cell body. Currently, high-resolution structures are only available for smaller IFT-B subcomplexes leaving > 50% structurally uncharacterized. Here, we used Alphafold to structurally model the 15-subunit IFT-B complex. The model was validated using cross-linking/mass-spectrometry data on reconstituted IFT-B complexes, X-ray scattering in solution, diffraction from crystals as well as site-directed mutagenesis and protein-binding assays. The IFT-B structure reveals an elongated and highly flexible complex consistent with cryo-electron tomographic reconstructions of IFT trains. The IFT-B complex organizes into IFT-B1 and IFT-B2 parts with binding sites for ciliary cargo and the inactive IFT dynein motor, respectively. Interestingly, our results are consistent with two different binding sites for IFT81/74 on IFT88/70/52/46 suggesting the possibility of different structural architectures for the IFT-B1 complex. Our data present a structural framework to understand IFT-B complex assembly, function, and ciliopathy variants.

中文翻译：

---

经生化验证的 15 亚基鞭毛内转运复合物 IFT-B 的结构模型


纤毛是普遍存在的真核细胞器，对于细胞运动、信号传导和感觉接收至关重要。纤毛的形成需要结构和信号成分的鞭毛内运输，并涉及组织成鞭毛内运输 (IFT) 复合物 IFT-A 和 IFT-B 的 22 种不同蛋白质，由分子马达运输。 IFT-B 复合体构成了聚合物 IFT 列车的主干，在纤毛和细胞体之间运输货物。目前，高分辨率结构仅适用于较小的 IFT-B 子复合物，而 > 50% 的结构未表征。在这里，我们使用 Alphafold 对 15 个亚基 IFT-B 复合物进行结构建模。使用重构的 IFT-B 复合物的交联/质谱数据、溶液中的 X 射线散射、晶体衍射以及定点诱变和蛋白质结合测定来验证该模型。 IFT-B 结构揭示了一种细长且高度灵活的复合体，与 IFT 列车的冷冻电子断层扫描重建一致。 IFT-B 复合物组织成 IFT-B1 和 IFT-B2 部分，分别具有纤毛货物和非活性 IFT 动力蛋白马达的结合位点。有趣的是，我们的结果与 IFT88/70/52/46 上 IFT81/74 的两个不同结合位点一致，表明 IFT-B1 复合物可能存在不同的结构体系。我们的数据提供了一个结构框架来理解 IFT-B 复合物的组装、功能和纤毛病变异。