Mercier Pascal, Lewis Michael J, Hau D Duong, Saltibus Linda F, Xiao Wei, Spyracopoulos Leo
Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Protein Sci. 2007 Apr;16(4):602-14. doi: 10.1110/ps.062358007. Epub 2007 Feb 27.
A key step in the signaling cascade responsible for activation of the transcription factor NF-kappaB involves Lys63-linked polyubiquitination of TRAF6. Covalent attachment of ubiquitin (Ub) to TRAF6, and subsequent poly(Ub) chain synthesis, is catalyzed by the hUev1a-hUbc13 heterodimer. hUbc13 is a catalytically competent E2 enzyme, and hUev1a is an E2-like protein that binds substrate Ub. The hUev1a-hUbc13 heterodimer is targeted to TRAF6 through interactions between hUbc13 and the N-terminal RING domain from TRAF6. Nuclear magnetic resonance (NMR) spectroscopy was used to determine the solution state structure of the RING domain from human TRAF6, and the interaction between hUbc13 and TRAF6 was characterized using NMR chemical shift mapping. The main-chain dynamics of the RING domain from TRAF6 were studied using (15)N NMR relaxation. Analysis of the main-chain dynamics data indicates that residues within the alpha-helix and beta-sheet of the RING domain are as rigid as regions of canonical secondary structure in larger proteins, consistent with the biological role of RING-domain E3 proteins, which requires that the E3 contain a recognition site for recruitment of E2 ubiquitin conjugation enzymes.
负责激活转录因子NF-κB的信号级联反应中的关键一步涉及TRAF6的赖氨酸63连接的多聚泛素化。泛素（Ub）与TRAF6的共价连接以及随后的多聚（Ub）链合成由hUev1a-hUbc13异二聚体催化。hUbc13是一种具有催化活性的E2酶，hUev1a是一种结合底物Ub的类E2蛋白。hUev1a-hUbc13异二聚体通过hUbc13与TRAF6的N端RING结构域之间的相互作用靶向TRAF6。利用核磁共振（NMR）光谱确定人TRAF6的RING结构域的溶液状态结构，并使用NMR化学位移映射表征hUbc13与TRAF6之间的相互作用。利用（15）N NMR弛豫研究TRAF6的RING结构域的主链动力学。主链动力学数据分析表明，RING结构域的α-螺旋和β-折叠内的残基与较大蛋白质中典型二级结构区域一样刚性，这与RING结构域E3蛋白的生物学作用一致，即E3需要包含一个用于招募E2泛素缀合酶的识别位点。
