【作者】 张勇；

【导师】 田长麟；

【作者基本信息】 中国科学技术大学 ， 生化与分子生物学， 2025， 博士

【摘要】 Kv1.3是一种在免疫细胞中大量表达的电压门控钾离子通道,在免疫应答过程中发挥重要作用。该通道通过参与调节T细胞激活过程中的胞内钙信号,从而诱导T细胞激活、增殖及细胞因子分泌。因此,Kv1.3抑制剂被认为是治疗自身免疫病的极具潜力的候选药物。天然毒素多肽是Kv1.3抑制剂的重要分子来源,多肽比小分子具有更大的相互作用表面积,使其在亚型选择性上更具优势,在靶向Kv1.3的抑制剂开发中展现出巨大潜力。尽管对于Kv1.3特异性抑制剂已经进行了大量研究,但目前尚无相关药物成功上市。造成这一现状的主要原因包括:Kv1.3自身及其与抑制剂复合物的高分辨结构尚未解析,研究者难以基于结构信息设计并改造出兼具高选择性和良好安全性的药物。并且Kv1.3通道具有C型失活特性,失活过程中发生的构象变化可能影响配体结合位点,从而增大药物设计的复杂性。此外,离子通道本身属于难以通过高通量方法研究的靶标,当前针对离子通道潜在药物的高通量筛选方法仍存在一些局限,这也制约了Kv1.3抑制剂的高效发现与开发,因此相关筛选方法的优化与创新亟待推进。在本文中,通过单颗粒冷冻电镜技术（Cryo-EM）解析了单独的Kv1.3通道结构（Apo-Kv1.3,3.2?）以及Kv1.3与多肽抑制剂Od K2的复合物的结构（Fab-Od K2-Kv1.3,2.67?）。两种结构整体较为相似,电压感受域（VSD）和门控域均处于激活状态:VSD区的S4螺旋上移,门控区打开,直径可允许水合钾离子通过。两者的主要差异在选择性过滤器区域,在Apo-Kv1.3的选择性过滤器中并未观察到S2位点的钾离子密度,且维持此区域稳定的氢键网络已经破坏,这是钾通道失活状态的典型结构特征。所以,该结构可能代表了一种介于激活态与C型失活态之间的中间构象。而在Fab-Od K2-Kv1.3结构中,择性过滤器区域的S2至S4位点均有到钾离子结合,S1位点由于Od K2中K27残基侧链与选择性过滤器的直接相互作用,无法结合钾离子。此外,选择性过滤器区域形成了稳定的氢键网络,进一步提示Od K2的结合可能使Kv1.3通道稳定于激活构象。Od K2结合在Kv1.3通道跨膜区的胞外侧,位于选择性过滤器上方,与通道外孔区残基形成广泛相互作用。其中,Od K2的K27位点残基侧链直接插入外孔的选择性过滤器,与Kv1.3的Y447残基的主链羰基氧原子形成配位,以堵孔的方式阻塞了钾离子传导路径。结合结构分析与电生理实验验证,进一步明确了Od K2与Kv1.3之间的关键相互作用残基对。为实现对靶向Kv1.3多肽配体的高效直观筛选,发展了一种基于¹⁹F-NMR化学位移编码的多肽配体筛选方法。可以同时对多种多肽进行筛选,在一维?¹⁹F-NMR谱图中即可直接识别出对Kv1.3具有亲和性的多肽。该策略为膜蛋白类药物靶点（如离子通道、G蛋白偶联受体GPCRs、转运体等）提供了一种简便、可靠的多肽配体筛选工具,具有广泛的应用前景。综上所述,本文通过单颗粒冷冻电镜技术解析了Apo-Kv1.3和Fab-Od K2-Kv1.3复合物的结构,填补了Kv1.3靶向药物研发中结构信息的空白,为多肽类抑制剂的理性设计与优化提供了重要的理论依据。并且解析的Kv1.3通道结构分别处于不同的状态,为深入理解其C型失活机制及开展状态依赖性配体设计提供了结构基础。同时,我们建立了一种基于¹⁹F-NMR化学位移编码的多肽配体筛选方法,能够高效识别与Kv1.3具有亲和力的候选分子。该方法同样适用于其他膜蛋白类药物靶点的配体筛选,具有广泛的应用潜力,有望加速多肽药物的开发进程。更多还原

【Abstract】 Kv1.3 is a voltage-gated potassium channel that is abundantly expressed in immune cells and plays a crucial role in immune responses.By participating in the regulation of intracellular calcium signaling during T cell activation,Kv1.3 promotes T cell activation,proliferation,and cytokine secretion.Therefore,Kv1.3 inhibitors are considered highly promising candidates for the treatment of autoimmune diseases.Natural toxin-derived peptides are an important source of Kv1.3 inhibitors.Compared with small molecules,peptides offer a larger interaction surface area,giving them an advantage in subtype selectivity,and have demonstrated great potential in the development of Kv1.3-targeted inhibitors.Despite extensive research on specific Kv1.3inhibitors,no related drug has yet reached the market.The main reasons for this include:the high-resolution structures of Kv1.3 and its inhibitor-bound complexes have not been resolved,making it difficult for researchers to design and modify drugs with both high selectivity and good safety profiles based on structural information.Moreover,Kv1.3channels exhibit C-type inactivation,and the conformational changes occurring during inactivation may affect ligand-binding sites,thereby increasing the complexity of drug design.In addition,ion channels are inherently challenging targets for high-throughput approaches,and the current methods for high-throughput screening of potential ion channel drugs still have limitations.This has hindered the efficient discovery and development of Kv1.3 inhibitors,highlighting the urgent need for optimization and innovation in relevant screening strategies.In this study,we determined the structures of the Kv1.3 channel alone（Apo-Kv1.3,3.2?）and in complex with the peptide inhibitor Od K2（Fab-Od K2-Kv1.3,2.67?）using single-particle cryo-electron microscopy（Cryo-EM）.The two structures are overall similar,with both the voltage-sensing domain（VSD）and the gating domain in an activated state:the S4 helix of the VSD is in an upward position,and the activation gate is open to a diameter sufficient for hydrated potassium ions to pass through.The primary difference lies in the selectivity filter region.In Apo-Kv1.3,no potassium ion density is observed at the S2 site,and the hydrogen-bond network stabilizing this region is disrupted,which is a hallmark structural feature of channel inactivation.Thus,this structure may represent an intermediate conformation between the activated and C-type inactivated states.In contrast,in the Fab-Od K2-Kv1.3 structure,potassium ions are bound at the S2 to S4 sites,whereas the S1 site is unoccupied due to direct interaction between the side chain of the K27 residue in Od K2 and the selectivity filter.Furthermore,a stable hydrogen-bond network is present in the selectivity filter region,suggesting that Od K2 binding may stabilize Kv1.3 in an activated conformation.Od K2binds to the extracellular side of the transmembrane domain,directly above the selectivity filter,engaging in extensive interactions with residues in the channel’s outer pore region.Notably,the side chain of K27 in Od K2 penetrates into the selectivity filter of the outer pore and coordinates with the backbone carbonyl oxygen of Kv1.3 residue Y447,thereby occluding the ion conduction pathway in a pore-blocking manner.Structural analysis combined with electrophysiological validation further identified key interacting residues between Od K2 and Kv1.3.To enable efficient and intuitive screening of peptide ligands targeting Kv1.3,we developed a ¹⁹F-NMR chemical shift-encoded peptide ligand screening method.This approach allows simultaneous screening of multiple peptides and direct identification of those with affinity for Kv1.3 in a one-dimensional ¹⁹F-NMR spectrum.This strategy offers a simple and reliable tool for peptide ligand screening against membrane protein drug targets such as ion channels,G protein-coupled receptors（GPCRs）,and transporters,with broad application potential.In summary,we resolved the structures of Apo-Kv1.3 and the Fab-Od K2-Kv1.3complex using single-particle cryo-EM,filling an important gap in structural information relevant to Kv1.3 drug discovery and providing a solid theoretical basis for the rational design and optimization of peptide inhibitors.The Kv1.3 structures captured in distinct functional states also offer structural insights into its C-type inactivation mechanism and provide a foundation for the design of state-dependent ligands.Additionally,we established a ¹⁹F-NMR chemical shift-encoded peptide screening method capable of efficiently identifying candidate molecules with Kv1.3affinity.This method is equally applicable to other membrane protein drug targets and holds broad potential to accelerate peptide drug development.更多还原