Wang Jiaolong, Kidmose Rune T, Boegholm Niels, Zacharia Nevin K, Thomsen Mads B, Christensen Anni, Malik Tara, Lechtreck Karl, Lorentzen Esben
Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark.
Department of Cellular Biology, University of Georgia, Athens, Georgia, USA.
J Biol Chem. 2025 Mar;301(3):108237. doi: 10.1016/j.jbc.2025.108237. Epub 2025 Jan 27.
Outer dynein arms (ODAs) are essential for ciliary motility and are preassembled in the cytoplasm before trafficking into cilia by intraflagellar transport (IFT). ODA16 is a key adaptor protein that links ODAs to the IFT machinery via direct interaction with the IFT46 protein. However, the molecular mechanisms regulating the assembly, transport, and release of ODAs remain poorly understood. Here, we employ AlphaPulldown, an in silico screening method, to identify direct interactors of ODA16, including the dynein adaptor IDA3 and the small GTPase Arl3. We use structural modeling, biochemical, and biophysical assays on Chlamydomonas and human proteins to elucidate the interactions and regulatory mechanisms governing the IFT of ODAs. We identify a conserved N-terminal motif in Chlamydomonas IFT46 that mediates its binding to one side of the ODA16 structure. Biochemical dissection reveals that IDA3 and Arl3 bind to the same surface of ODA16 (the C-terminal β-propeller face), which is opposite to the IFT46 binding site, enabling them to dissociate ODA16 from IFT46, likely through an allosteric mechanism. Our findings provide mechanistic insights into the concerted actions of IFT and adaptor proteins in ODA transport and regulation.
外动力蛋白臂（ODAs）对于纤毛运动至关重要，并且在通过鞭毛内运输（IFT）进入纤毛之前在细胞质中预先组装。ODA16是一种关键的衔接蛋白，通过与IFT46蛋白直接相互作用将ODAs与IFT机制联系起来。然而，调节ODAs组装、运输和释放的分子机制仍知之甚少。在这里，我们采用计算机筛选方法AlphaPulldown来鉴定ODA16的直接相互作用蛋白，包括动力蛋白衔接蛋白IDA3和小GTP酶Arl3。我们对衣藻和人类蛋白质进行结构建模、生化和生物物理分析，以阐明控制ODAs的IFT的相互作用和调节机制。我们在衣藻IFT46中鉴定出一个保守的N端基序，该基序介导其与ODA16结构的一侧结合。生化分析表明，IDA3和Arl3与ODA16的同一表面（C端β螺旋桨面）结合，该表面与IFT46结合位点相对，这可能使它们通过变构机制将ODA16与IFT46解离。我们的研究结果为IFT和衔接蛋白在ODA运输和调节中的协同作用提供了机制上的见解。
