Huntington's disease is characterised by the CAG expansion in the huntingtin gene above a critical threshold of ~35 repeats, which results in the polyglutamine expansion of the huntingtin protein (HTT). The biological roles of wildtype HTT and the associated mechanisms of disease pathology caused by expanded HTT remain incompletely understood, in part due to challenges in directly characterising interactions between HTT and putative binding partners. Here we describe a biochemical toolkit of two rationally designed, high-quality recombinant HTT subdomains; one consisting of the N-terminal HEAT region and bridge domain (NTD) and the second comprising the C-terminal HEAT domain (CTD). Using biophysical methods and cryo-electron microscopy, we show that these smaller subdomains are natively folded and can associate to reconstitute a functional full-length HTT structure capable of forming a near native-like complex with the 40 kDa HTT-associated protein (HAP40). We also report biotin-tagged variants of these subdomains, as well as full-length HTT, that permit immobilisation of each protein for quantitative biophysical assays without impacting protein quality. We demonstrate that the CTD alone can form a stable complex when co-expressed with HAP40, which can be structurally resolved. The CTD/HAP40 complex binds to the NTD, with a dissociation constant of approximately 10 nM as measured by bio-layer interferometry. We validated the interaction between the CTD and HAP40 in cells using a luciferase two-hybrid assay and used individual subdomain constructs to demonstrate their respective stabilization of HAP40 in cells. These open-source biochemical tools will prove useful to the wider HD community for the study of fundamental HTT biology, discover new macromolecular or small-molecule binding partners and mapping of their interaction sites across this very large protein.

中文翻译：

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扩大亨廷顿病研究工具箱；用于亨廷顿蛋白生化和结构研究的经过验证的子域蛋白质构建体

亨廷顿氏病的特征是亨廷顿基因中的 CAG 扩增超过约 35 次重复的临界阈值，这导致亨廷顿蛋白 (HTT) 的聚谷氨酰胺扩增。野生型 HTT 的生物学作用以及由扩展的 HTT 引起的疾病病理学的相关机制仍未完全了解，部分原因是直接表征 HTT 和假定的结合伙伴之间的相互作用存在挑战。在这里，我们描述了两个合理设计的高质量重组 HTT 子域的生化工具包；一个由 N 端 HEAT 区和桥域 (NTD) 组成，第二个由 C 端 HEAT 区 (CTD) 组成。使用生物物理方法和冷冻电子显微镜，我们表明这些较小的子域是天然折叠的并且可以关联以重建功能性全长 HTT 结构，该结构能够与 40 kDa HTT 相关蛋白 (HAP40) 形成接近天然的复合物。我们还报告了这些子域的生物素标记变体，以及全长 HTT，它们允许固定每种蛋白质以进行定量生物物理测定，而不会影响蛋白质质量。我们证明，当与 HAP40 共表达时，单独的 CTD 可以形成一个稳定的复合体，这可以在结构上得到解决。CTD/HAP40 复合物与 NTD 结合，通过生物层干涉测量法测量解离常数约为 10 nM。我们使用荧光素酶双杂交试验验证了细胞中 CTD 和 HAP40 之间的相互作用，并使用单独的子域结构来证明它们各自对细胞中 HAP40 的稳定性。这些开源生​​化工具将证明对更广泛的 HD 社区有用，可用于研究基础 HTT 生物学、发现新的大分子或小分子结合伙伴以及绘制它们在这种非常大的蛋白质上的相互作用位点。