PDF(Pubmed)
Abstract：
KEAP1 promotes the ubiquitin-dependent degradation of NRF2 by assembling into a CUL3-dependent ubiquitin ligase complex. Oxidative and electrophilic stress inhibit KEAP1 allowing NRF2 to accumulate for the transactivation of stress response genes. To date there are no structures of the KEAP1-CUL3 interaction nor binding data to show the contributions of different domains to their binding affinity. We determined a crystal structure of the BTB and 3-box domains of human KEAP1 in complex with the CUL3 N-terminal domain that showed a heterotetrameric assembly with 2:2 stoichiometry. To support the structural data, we developed a versatile TR-FRET-based assay system to profile the binding of BTB-domain-containing proteins to CUL3 and determine the contribution of distinct protein features, revealing the importance of the CUL3 N-terminal extension for high affinity binding. We further provide direct evidence that the investigational drug CDDO does not disrupt the KEAP1-CUL3 interaction, even at high concentrations, but reduces the affinity of KEAP1-CUL3 binding. The TR-FRET-based assay system offers a generalizable platform for profiling this protein class and may form a suitable screening platform for ligands that disrupt these interactions by targeting the BTB or 3-box domains to block E3 ligase function.
摘要：
KEAP1通过组装成CUL3依赖性泛素连接酶复合物来促进NRF2的泛素依赖性降解。氧化和亲电胁迫抑制KEAP1，从而使NRF2积累以进行应激反应基因的反式激活。迄今为止,没有KEAP1-CUL3相互作用的结构或结合数据来显示不同结构域对其结合亲和力的贡献。我们确定了人KEAP1的BTB和3盒结构域与CUL3N末端结构域复合的晶体结构，该结构域显示出2：2化学计量的异四聚体组装。为了支持结构数据，我们开发了一种基于TR-FRET的多功能测定系统，以描述含BTB结构域的蛋白质与CUL3的结合，并确定不同蛋白质特征的贡献。揭示了CUL3N端延伸对高亲和力结合的重要性。我们进一步提供了直接证据，表明研究药物CDDO不会破坏KEAP1-CUL3相互作用，即使在高浓度下，但降低了KEAP1-CUL3结合的亲和力。基于TR-FRET的测定系统提供了用于分析该蛋白质类别的可通用平台,并且可以形成用于配体的合适筛选平台,所述配体通过靶向BTB或3盒结构域以阻断E3连接酶功能来破坏这些相互作用。
