Tryptophan mutations in G3BP1 tune the stability of a cellular signaling hub by weakening transient interactions with Caprin1 and USP10.
Tryptophan mutations in G3BP1 tune the stability of a cellular signaling hub by weakening transient interactions with Caprin1 and USP10.
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DOI：
10.1016/j.jbc.2022.102552
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发表时间：
2022-12
影响因子：
4.8
通讯作者：
Madden, Dean R.
中科院分区：
文献类型：
--
作者：
Sheehan, Colin T.;Hampton, Thomas H.;Madden, Dean R.
内含子无序蛋白（IDP）通常与多种蛋白质协调瞬时相互作用，以介导大型蛋白质网络中的复杂信号。其中，IDP枢纽蛋白G3 BP 1可以与细胞质磷蛋白Caprin 1和泛素肽酶USP 10形成复合物;对USP 10活性的控制有助于致病性毒力系统，该系统靶向离子通道CFTR的内吞再循环。然而，虽然已知许多IDP枢纽蛋白的蛋白质相互作用物的身份，但成对亲和力与蛋白质募集和活性程度之间的关系尚不清楚。在这里，我们描述了这些G3 BP 1亲和力的体外分析，并显示特定G3 BP 1残基的色氨酸取代降低了其对USP 10和Caprin 1的亲和力。我们发现，这些相同的突变降低了全长蛋白质之间的复合物的稳定性，这表明共纯化可以作为相互作用强度的替代措施。G3 BP 1 TripleW（F15 W/F33 W/F124 W）突变体的晶体结构显示W33侧链的明显重定向，与USP 10和Caprin 1产生空间冲突。此外，USP 10和Caprin 1肽的氨基酸扫描揭示了核心基序中残基取代能力的相似性和差异，以及具有产生更高亲和力肽潜力的特异性取代。总之，这些数据表明，组件结合亲和力的微小变化可能对细胞相互作用中心的组成产生显著影响。这些特定的蛋白质突变可以用来操纵复杂的蛋白质网络，为未来研究这些网络在细胞过程中的作用提供信息。
Intrinsically disordered proteins (IDPs) often coordinate transient interactions with multiple proteins to mediate complex signals within large protein networks. Among these, the IDP hub protein G3BP1 can form complexes with cytoplasmic phosphoprotein Caprin1 and ubiquitin peptidase USP10; the resulting control of USP10 activity contributes to a pathogenic virulence system that targets endocytic recycling of the ion channel CFTR. However, while the identities of protein interactors are known for many IDP hub proteins, the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood. Here, we describe in vitro analysis of these G3BP1 affinities and show tryptophan substitutions of specific G3BP1 residues reduce its affinity for both USP10 and Caprin1. We show that these same mutations reduce the stability of complexes between the full-length proteins, suggesting that copurification can serve as a surrogate measure of interaction strength. The crystal structure of G3BP1 TripleW (F15W/F33W/F124W) mutant reveals a clear reorientation of the side chain of W33, creating a steric clash with USP10 and Caprin1. Furthermore, an amino-acid scan of USP10 and Caprin1 peptides reveals similarities and differences in the ability to substitute residues in the core motifs as well as specific substitutions with the potential to create higher affinity peptides. Taken together, these data show that small changes in component binding affinities can have significant effects on the composition of cellular interaction hubs. These specific protein mutations can be harnessed to manipulate complex protein networks, informing future investigations into roles of these networks in cellular processes.
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影响因子：
4.8
作者：
Cristea, Ileana M.;Carroll, John-William N.;MacDonald, Margaret R.
通讯作者：
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影响因子：
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通讯作者：
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影响因子：
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通讯作者：
Madden, Dean R.
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作者：
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通讯作者：
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