Lys63-linked ubiquitin chain adopts multiple conformational states for specific target recognition.
Lys63-linked ubiquitin chain adopts multiple conformational states for specific target recognition.
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作者：
Liu Z;Gong Z;Jiang WX;Yang J;Zhu WK;Guo DC;Zhang WP;Liu ML;Tang C
多泛素由多个共价连接的泛素组成，可识别无数靶标。多泛素是自由的或与配体结合的，在不同的泛素亚基排列中发现。为了解多泛素四级可塑性的结构基础，探索靶识别机制，我们对Lys63-linked diubiquitin （K63-Ub2）的构象空间进行了表征。根据亚基间顺磁核磁共振数据，我们发现自由的K63-Ub2作为一个由多个闭合和开放的季态组成的动态系综存在。四元动力学使得K63-Ub2在多种信号通路中被特异性识别。当与目标蛋白结合时，其中一个预先存在的四元态被选择和稳定。一个点突变改变了不同状态之间的平衡，调节了对K63-Ub2配体的结合亲和力。这种在四元水平上的构象选择机制可能被不同长度和连接的多泛素用于目标识别。DOI: http://dx.doi.org/10.7554/eLife.05767.001蛋白质可以用其他分子标记，指示细胞应该如何处理该蛋白质。例如，被称为泛素的小蛋白质标记的蛋白质——与其他泛素分子连接形成“多泛素”——可能会在细胞内被破坏或重新定位。像所有的蛋白质一样，泛素是由氨基酸链组成的。特定的氨基酸在单个泛素之间形成键，形成多泛素，这些键的性质影响多泛素对标记蛋白的作用。一个连锁涉及63位的赖氨酸氨基酸（称为Lys63）。这种联系是在多泛素中发现的，多泛素参与修复受损蛋白质并将目标蛋白质重新定位到细胞的一部分，在那里它们被用于免疫反应。为了发挥这些不同的作用，多泛素必须能够区分各种靶蛋白。蛋白质的形状决定了它的工作方式，大多数蛋白质在不同的形状之间不断快速地转换。先前的研究表明，lys63连接的多泛素本身只能呈现一个细长的“开放”结构。目前尚不清楚这种蛋白质是否能在不首先与靶蛋白结合的情况下呈现紧凑的“封闭”结构。Liu等人使用了一种被称为核磁共振（NMR）的技术来探索lys63连接的泛素链在不与其他蛋白质结合时的高分辨率结构。结果表明，该蛋白有很大比例处于封闭状态，且至少存在一种开放形态和两种封闭形态。Liu等人认为，未结合的lys63连接的泛素链的形状决定了哪些其他蛋白质可以被结合，并且这种结合稳定了泛素的形状。这种结合机制被称为构象选择。需要进一步的工作来分析其他多泛素链是否以类似的方式识别它们的伴侣。DOI: http://dx.doi.org/10.7554/eLife.05767.002
A polyubiquitin comprises multiple covalently linked ubiquitins and recognizes myriad targets. Free or bound to ligands, polyubiquitins are found in different arrangements of ubiquitin subunits. To understand the structural basis for polyubiquitin quaternary plasticity and to explore the target recognition mechanism, we characterize the conformational space of Lys63-linked diubiquitin (K63-Ub2). Refining against inter-subunit paramagnetic NMR data, we show that free K63-Ub2 exists as a dynamic ensemble comprising multiple closed and open quaternary states. The quaternary dynamics enables K63-Ub2 to be specifically recognized in a variety of signaling pathways. When binding to a target protein, one of the preexisting quaternary states is selected and stabilized. A point mutation that shifts the equilibrium between the different states modulates the binding affinities towards K63-Ub2 ligands. This conformational selection mechanism at the quaternary level may be used by polyubiquitins of different lengths and linkages for target recognition. DOI: http://dx.doi.org/10.7554/eLife.05767.001 Proteins can be tagged with other molecules that indicate what the cell should do with that protein. For example, proteins tagged with a small protein called ubiquitin—which is linked to other ubiquitin molecules to form ‘polyubiquitin’—may be destroyed or relocated within a cell. Like all proteins, a ubiquitin is made up of chains of amino acids. Specific amino acids form the linkages between individual ubiquitins to form a polyubiquitin, and the nature of these linkages influences the effect that a polyubiquitin has on the tagged protein. One linkage involves a lysine amino acid at position 63 (known as Lys63). This linkage is found in the polyubiquitin that is involved in repairing damaged proteins and relocating target proteins to a part of the cell where they are utilized for immune response. To perform these different roles, the polyubiquitin must be able to distinguish between a variety of target proteins. The shape that a protein takes on determines how it works, and most proteins constantly and rapidly switch between different shapes. Previous work suggested that the Lys63-linked polyubiquitin could only take on an elongated ‘open’ structure by itself. It was not clear whether the protein could take on a compact ‘closed’ structure without first binding to a target protein. Liu et al. used a technique known as nuclear magnetic resonance (NMR) to explore the high-resolution structures of the Lys63-linked ubiquitin chain when they are not bound to other proteins. The results showed that a large percentage of the protein was in a closed state, and that there were at least one open shape and two kinds of closed shapes. Liu et al. suggest that the shape of the unbound Lys63-linked ubiquitin chain determines what other proteins can be bound, and that the binding stabilizes the shape of the ubiquitin. This mechanism of binding is known as conformational selection. Further work is required to analyze whether other polyubiquitin chains recognize their partners in a similar manner. DOI: http://dx.doi.org/10.7554/eLife.05767.002