The modular structure of SIP facilitates its role in stabilizing multiprotein assemblies
The modular structure of SIP facilitates its role in stabilizing multiprotein assemblies
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DOI：
10.1021/bi0502689
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发表时间：
2005-07-12
期刊：
影响因子：
2.9
通讯作者：
Chazin, WJ
中科院分区：
文献类型：
--
作者：
Bhattacharya, S;Lee, YT;Chazin, WJ
siah相互作用蛋白（SIP）被鉴定为一种新的适配器，它将Siah-1的E3泛素连接酶活性与Skp1和Ebi F-Box蛋白在TCF/LEF基因的转录激活因子β -连环蛋白的降解中物理连接起来。在这项研究中，我们利用溶液核磁共振光谱表征了SIP的结构域结构，其中包括在n端灵活连接CS结构域的新型螺旋发夹结构域和非结构化羧基末端SGS结构域。通过将Siah-1和Skp1等功能重要的蛋白-蛋白相互作用位点映射到SIP的单个结构域，这些研究得到了补充。基于核磁共振的化学位移摄动分析表明Siah-1与SIP N和CS结构域之间的柔性连接物相互作用。连接体中的这个相互作用位点不会干扰n端结构区域中的残基，但在全长蛋白的情况下，它似乎限制了SIP CS结构域的旋转自由。相反，Skp1只通过弱相互作用参与SIP CS域，而不与其他域耦合。SIP模块化结构的主要作用似乎是将这两种蛋白质引入物理接近，并在完整的scf型复合体中协调β -连环蛋白多泛素化所需的方向。
Siah-interacting protein (SIP) was identified as a novel adaptor that physically links the E3 ubiquitin ligase activity of Siah-1 with Skp1 land Ebi F-Box protein in the degradation of beta-catenin, a transcriptional activator of TCF/LEF genes. In this study, we have used solution NMR spectroscopy to characterize the domain structure of SIP, which includes a novel helical hairpin domain at the N-terminus flexibly linked to a CS domain and an unstructured carboxy terminal SGS domain. These studies have been complemented by mapping the sites of functionally important protein-protein interactions involving Siah-1 and Skp1 to individual domains of SIP. NMR-based chemical shift perturbation assays show that Siah-1 interacts with the flexible linker between SIP N and CS domains. This site for interaction in the linker does not perturb residues in the structured region at the N-terminus but does appear to restrict the rotational freedom of the SIP CS domain in the context of the full-length protein. In contrast, Skp1 engages the SIP CS domain exclusively through weak interactions that are not coupled to the other domains. The principal role of the modular structure of SIP appears to be in bringing these two proteins into physical proximity and orchestrating the orientation required for polyubiquitination of beta-catenin in the intact SCF-type complex.