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Reconstitution and Structural Analysis of a HECT Ligase-Ubiquitin Complex via an Activity-Based Probe
ACS Chemical Biology ( IF 4 ) Pub Date : 2021-08-17 , DOI: 10.1021/acschembio.1c00433
Rahul M Nair 1 , Ayshwarya Seenivasan 2 , Bing Liu 1 , Dan Chen 1 , Edward D Lowe 3 , Sonja Lorenz 2
Affiliation  

Ubiquitin activity-based probes have proven invaluable in elucidating structural mechanisms in the ubiquitin system by stabilizing transient macromolecular complexes of deubiquitinases, ubiquitin-activating enzymes, and the assemblies of ubiquitin-conjugating enzymes with ubiquitin ligases of the RING-Between-RING and RING-Cysteine-Relay families. Here, we demonstrate that an activity-based probe, ubiquitin-propargylamine, allows for the preparative reconstitution and structural analysis of the interactions between ubiquitin and certain HECT ligases. We present a crystal structure of the ubiquitin-linked HECT domain of HUWE1 that defines a catalytically critical conformation of the C-terminal tail of the ligase for the transfer of ubiquitin to an acceptor protein. Moreover, we observe that ubiquitin-propargylamine displays selectivity among HECT domains, thus corroborating the notion that activity-based probes may provide entry points for the development of specific, active site-directed inhibitors and reporters of HECT ligase activities.

中文翻译:

通过基于活性的探针重构和结构分析 HECT 连接酶-泛素复合物

基于泛素活性的探针通过稳定去泛素化酶、泛素激活酶的瞬时大分子复合物以及泛素缀合酶与 RING-Between-RING 和 RING-半胱氨酸中继家族。在这里,我们证明了一种基于活性的探针,泛素-炔丙胺,允许对泛素和某些 HECT 连接酶之间的相互作用进行制备性重组和结构分析。我们展示了 HUWE1 泛素连接 HECT 域的晶体结构,该结构定义了连接酶 C 末端尾部的催化关键构象,用于将泛素转移到受体蛋白。而且,
更新日期:2021-09-17
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