Structural Biology and NMR Laboratory, University of Copenhagen, Copenhagen N, Denmark.
REPIN, University of Copenhagen, Copenhagen N, Denmark.
Protein Sci. 2023 Sep;32(9):e4733. doi: 10.1002/pro.4733.
Intrinsically disordered proteins (IDPs) are often multifunctional and frequently posttranslationally modified. Deleted in split hand/split foot 1 (Dss1-Sem1 in budding yeast) is a highly multifunctional IDP associated with a range of protein complexes. However, it remains unknown if the different functions relate to different modified states. In this work, we show that Schizosaccharomyces pombe Dss1 is a substrate for casein kinase 2 in vitro, and we identify three phosphorylated threonines in its linker region separating two known disordered ubiquitin-binding motifs. Phosphorylations of the threonines had no effect on ubiquitin-binding but caused a slight destabilization of the C-terminal α-helix and mediated a direct interaction with the forkhead-associated (FHA) domain of the RING-FHA E3-ubiquitin ligase defective in mitosis 1 (Dma1). The phosphorylation sites are not conserved and are absent in human Dss1. Sequence analyses revealed that the Txx(E/D) motif, which is important for phosphorylation and Dma1 binding, is not linked to certain branches of the evolutionary tree. Instead, we find that the motif appears randomly, supporting the mechanism of ex nihilo evolution of novel motifs. In support of this, other threonine-based motifs, although frequent, are nonconserved in the linker, pointing to additional functions connected to this region. We suggest that Dss1 acts as an adaptor protein that docks to Dma1 via the phosphorylated FHA-binding motifs, while the C-terminal α-helix is free to bind mitotic septins, thereby stabilizing the complex. The presence of Txx(D/E) motifs in the disordered regions of certain septin subunits may be of further relevance to the formation and stabilization of these complexes.
无规则蛋白（IDPs）通常具有多功能性，并经常发生翻译后修饰。分裂手/足 1 缺失蛋白（ budding 酵母中的 Dss1-Sem1）是一种高度多功能的 IDP，与多种蛋白质复合物有关。然而，不同的功能是否与不同的修饰状态有关尚不清楚。在这项工作中，我们表明，酿酒酵母的 Dss1 是体外酪蛋白激酶 2 的底物，并且我们在其连接区中鉴定出三个磷酸化苏氨酸，该连接区分隔两个已知的无规则泛素结合基序。这些苏氨酸的磷酸化对泛素结合没有影响，但导致 C 末端α-螺旋的轻微不稳定性，并介导与有丝分裂缺陷 1（Dma1）的叉头相关（FHA）域的直接相互作用。这些磷酸化位点在人类 Dss1 中没有保守性，也不存在。序列分析表明，对磷酸化和 Dma1 结合很重要的 Txx（E/D）基序与进化树的某些分支没有联系。相反，我们发现该基序随机出现，支持新基序从无到有的进化机制。支持这一观点的是，尽管频繁出现，但其他基于苏氨酸的基序在连接区中没有保守性，这表明与该区域相关的其他功能。我们认为，Dss1 作为一种衔接蛋白，通过磷酸化 FHA 结合基序与 Dma1 对接，而 C 末端α-螺旋则自由结合有丝分裂隔蛋白，从而稳定复合物。某些隔蛋白亚基的无规则区中存在 Txx（D/E）基序可能与这些复合物的形成和稳定有关。
