Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, UK.
J Mol Biol. 2010 Nov 12;403(5):706-22. doi: 10.1016/j.jmb.2010.07.045. Epub 2010 Aug 4.
The architectural DNA-binding protein HMGB1 consists of two tandem HMG-box domains joined by a basic linker to a C-terminal acidic tail, which negatively regulates HMGB1-DNA interactions by binding intramolecularly to the DNA-binding faces of both basic HMG boxes. Here we demonstrate, using NMR chemical-shift mapping at different salt concentrations, that the tail has a higher affinity for the B box and that A box-tail interactions are preferentially disrupted. Previously, we proposed a model in which the boxes are brought together in a collapsed, tail-mediated assembly, which is in dynamic equilibrium with a more extended form. Small-angle X-ray scattering data are consistent with such a dynamic equilibrium between collapsed and extended structures and are best represented by an ensemble. The ensembles contain a significantly higher proportion of collapsed structures when the tail is present. (15)N NMR relaxation measurements show that full-length HMGB1 has a significantly lower rate of rotational diffusion than the tail-less protein, consistent with the loss of independent domain motions in an assembled complex. Mapping studies using the paramagnetic spin label MTSL [(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidin-3-yl)methyl methanethiosulfonate] placed at three locations in the tail confirm our previous findings that the tail binds to both boxes with some degree of specificity. The end of the tail lies further from the body of the protein and is therefore potentially free to interact with other proteins. MTSL labelling at a single site in the A domain (C44) causes detectable relaxation enhancements of B domain residues, suggesting the existence of a "sandwich"-like collapsed structure in which the tail enables the close approach of the basic domains. These intramolecular interactions are presumably important for the dynamic association of HMGB1 with chromatin and provide a mechanism by which protein-protein interactions or posttranslational modifications might regulate the function of the protein at particular sites, or at particular stages in the cell cycle.
结构域 DNA 结合蛋白 HMGB1 由两个串联的 HMG 盒结构域组成，通过碱性连接子与 C 端酸性尾巴相连，通过分子内结合到两个碱性 HMG 盒的 DNA 结合面上，负调控 HMGB1-DNA 相互作用。在这里，我们使用不同盐浓度下的 NMR 化学位移图谱证明，尾巴与 B 盒的亲和力更高，并且 A 盒-尾巴相互作用优先被破坏。以前，我们提出了一个模型，其中盒子在尾巴介导的组装中聚集在一起，该组装与更扩展的形式处于动态平衡。小角度 X 射线散射数据与这种折叠和扩展结构之间的动态平衡一致，并且最好由集合表示。当尾巴存在时，集合中包含更高比例的折叠结构。(15)N NMR 弛豫测量表明，全长 HMGB1 的旋转扩散率明显低于无尾蛋白，这与组装复合物中独立结构域运动的丧失一致。使用位于尾巴三个位置的顺磁自旋标记 MTSL [(1-氧代-2,2,5,5-四甲基-3-吡咯啉-3-基)甲基甲硫磺酸酯]进行的映射研究证实了我们以前的发现，即尾巴与两个盒子具有一定程度的特异性结合。尾巴的末端离蛋白质主体更远，因此可能可以自由与其他蛋白质相互作用。A 域 (C44) 中单个位置的 MTSL 标记会导致 B 域残基的可检测弛豫增强，这表明存在一种“三明治”折叠结构，其中尾巴使碱性结构域紧密接近。这些分子内相互作用对于 HMGB1 与染色质的动态缔合可能很重要，并为蛋白质-蛋白质相互作用或翻译后修饰可能在特定部位或细胞周期的特定阶段调节蛋白质功能提供了一种机制。
