Department of Chemistry, University of Cambridge , Lensfield Road, Cambridge CB2 1EW, United Kingdom.
Biochemistry. 2013 Aug 6;52(31):5236-46. doi: 10.1021/bi400758h. Epub 2013 Jul 25.
Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM-MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9-EloBC-Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM-MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems.
锚蛋白重复和 SOCS 盒（ASB）家族的蛋白作为 ECS 型（ElonginBC-Cullin-SOCS-box）Cullin RING E3 泛素连接酶（CRL）复合物的底物识别亚基，该复合物催化细胞蛋白的特异性多泛素化，将其靶向蛋白酶体降解。因此，ASB 多聚体复合物参与了许多细胞过程和途径；然而，它们的相互作用、组装和生物学功能仍知之甚少。为了增强我们对 ASB CRL 系统的理解，我们研究了由 ASB9、Elongin B、Elongin C（EloBC）和 Cullin 5 组成的四元多亚基复合物的结构、亲和力和组装。在这里，我们描述了几种生物物理技术的应用，包括差示扫描荧光法、等温滴定量热法（ITC）、纳米电喷雾电离和离子迁移质谱（IM-MS），为四元 ASB CRL 复合物提供结构和热力学信息。我们发现 ASB9 单独不稳定，但与 EloBC 形成稳定的三元复合物，该复合物与 Cullin 5 N 端结构域（Cul5NTD）具有高亲和力，但与 Cul2NTD 没有亲和力。单体 ASB9-EloBC-Cul5NTD 四元复合物的结构通过分子建模揭示，与 IM-MS 和温度依赖性 ITC 数据一致。这是第一个验证 ASB CRL 复合物四元 N 端组装结构信息的实验研究。结果表明，ASB E3 连接酶复合物以类似于其他 CRL 系统的方式发挥作用和组装，并为进一步研究这个重要的蛋白家族提供了一个分子研究平台。这里报道的数据也将有助于开发用于研究其他 ECS 型 CRL 系统的生物学功能和调节的化学探针。
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