Interplay of Disorder and Sequence Specificity in the Formation of Stable Dynein-Dynactin Complexes
Interplay of Disorder and Sequence Specificity in the Formation of Stable Dynein-Dynactin Complexes
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DOI：
10.1016/j.bpj.2020.07.023
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发表时间：
2020-09-01
影响因子：
3.4
通讯作者：
Barbar, Elisar
中科院分区：
文献类型：
--
作者：
Loening, Nikolaus M.;Saravanan, Sanjana;Barbar, Elisar
细胞质动力蛋白是一种真核动力蛋白复合物，沿着其调节蛋白动力蛋白，对细胞内细胞器的运输至关重要。动力蛋白与动力肌动蛋白的相互作用受动力蛋白的中间链（IC）亚基与动力肌动蛋白的p150（Glued）亚基之间的结合调节。尽管在这些蛋白质的大鼠版本中，这种相互作用主要涉及IC N-末端的单个α-螺旋区域，但在果蝇和酵母IC中，去除单个α-螺旋下游的新生螺旋（H2）显著降低了IC-p150（胶合的）复合物的稳定性。我们发现，对于来自不同物种的IC，在H2中的无序与其对结合亲和力的贡献之间存在相关性，并且在H2中的序列变化不改变无序水平显示出类似的结合行为。对嗜热毛壳菌IC的结构和相互作用的分析表明，C.嗜热菌IC具有低螺旋倾向，并确定H2直接结合p150的卷曲螺旋1B（CC 1B）结构域（Glued），从而解释了为什么H2对于紧密结合是必需的。等温滴定量热法，圆二色性，和NMR研究较小的CC 1B结构本地化的CC 1B区域最重要的一个紧密的相互作用与IC。这些结果表明，正是IC沿着其电荷的H2中的紊乱水平，而不是序列特异性，构成了其在启动紧密的IC-p150（胶合的）复合物形成中的重要性的基础。我们推测，新生的H2螺旋可能提供构象的灵活性，以启动绑定，而那些物种，有一个完全折叠的H2增选一个替代机制，促进p150（胶合）结合。
Cytoplasmic dynein is a eukaryotic motor protein complex that, along with its regulatory protein dynactin, is essential to the transport of organelles within cells. The interaction of dynein with dynactin is regulated by binding between the intermediate chain (IC) subunit of dynein and the p150(Glued) subunit of dynactin. Even though in the rat versions of these proteins this interaction primarily involves the single alpha-helix region at the N-terminus of the IC, in Drosophila and yeast ICs the removal of a nascent helix (H2) downstream of the single alpha-helix considerably diminishes IC-p150(Glued) complex stability. We find that for ICs from various species, there is a correlation between disorder in H2 and its contribution to binding affinity, and that sequence variations in H2 that do not change the level of disorder show similar binding behavior. Analysis of the structure and interactions of the IC from Chaetomium thermophilum demonstrates that the H2 region of C. thermophilum IC has a low helical propensity and establishes that H2 binds directly to the coiled-coil 1B (CC1B) domain of p150(Glued), thus explaining why H2 is necessary for tight binding. Isothermal titration calorimetry, circular dichroism, and NMR studies of smaller CC1B constructs localize the region of CC1B most essential for a tight interaction with IC. These results suggest that it is the level of disorder in H2 of IC along with its charge, rather than sequence specificity, that underlie its importance in initiating tight IC-p150(Glued) complex formation. We speculate that the nascent H2 helix may provide conformational flexibility to initiate binding, whereas those species that have a fully folded H2 have co-opted an alternative mechanism for promoting p150(Glued) binding.