A complex implicated in kidney health Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease that can lead to kidney failure. Mutations in the proteins PKD1 and PKD2 are linked to the disease, but the function of these proteins remains unclear, both in physiology and disease. PKD1 has been implicated in the sensing of chemical and mechanical force stimuli, and PKD2 is proposed to be a calcium ion channel. Su et al. show that the transmembrane regions form a PKD1-PKD2 complex assembled in a 1:3 ratio. Their high-resolution cryo–electron microscopy structure confirms that the complex adopts transient receptor potential channel architecture, with some distinctive features. Mapping disease-causing mutations onto the structure suggests that pathogenesis may come from incorrect folding or trafficking of the complex rather than from disruption of channel activity. Science, this issue p. eaat9819 This structure provides a framework for further investigations into a complex involved in polycystic kidney disease. INTRODUCTION Mutations in two genes, PKD1 and PKD2, are responsible for about 85 and 10% of all cases of autosomal dominant polycystic kidney disease (ADPKD), one of the most common monogenetic disorders. However, the physiological and pathophysiological functions of the gene products polycystin-1 and polycystin-2 (PC1 and PC2, also known as PKD1 and PKD2) are not well understood. PKD1, which comprises 4303 residues, may serve as a receptor that senses chemical and mechanical force stimuli, whereas PKD2, whose homotetrameric structure conforms to a typical group II transient receptor potential (TRP) channel, is hypothesized to be an endoplasmic reticulum Ca2+-release channel and regulate intracellular Ca2+ concentrations. The two proteins were predicted to coexist as a hetero-oligomer on primary cilia in the renal epithelium, although the molecular basis for the formation of this complex remains elusive. RATIONALE To investigate the assembly of PKD1 and PKD2, we sought to resolve the structure of the PKD1-PKD2 complex. After extensive screening for optimal constructs and expression systems, a homogeneous complex was obtained through coexpression of FLAG-tagged PKD1 and Twin-Strep–tagged PKD2 (hereafter referred to as PKD1 and PKD2 for simplicity). Approximately 100 μg of the complex was obtained through affinity purification and size exclusion chromatography from 40 to 50 liters of suspension human embryonic kidney (HEK) 293F cells. The structure of the complex was determined to 3.6-Å resolution with single-particle cryo–electron microscopy (cryo-EM). RESULTS PKD1 and PKD2 exhibit a 1:3 ratio in the structure. PKD1 consists of a voltage-gated ion channel (VGIC) fold that interacts with PKD2 to complete a domain-swapped TRP architecture. Several features, however, distinguish PKD1 from a canonical TRP channel. The S6 segment of PKD1 is broken in the middle, with the extracellular half, S6a, resembling pore helix 1 (PH1) in a typical VGIC. The sequence between S5 and S6a is highly flexible and disordered in the EM map. Three positively charged residues—Arg4100, Arg4107, and His4111—protrude into the putative ion-conducting path, likely impeding permeability of the Ca2+ ion. Therefore, the current structure may represent a potentially nonconductive state. A discretely folded domain, which contains five transmembrane helices (TMs) and a cytosolic PLAT (polycystin-1, lipoxygenase, and alpha toxin) domain, precedes the VGIC fold in PKD1. The extracellular TOP domain of PKD1, which is frequently targeted for mutations in ADPKD, deviates from the expected symmetric position by 15°, leaving a gap in the extracellular TOP ring. Compared to the homotypic interactions among PKD2 subunits, the weakened interface between PKD1 and PKD2 provides a clue to the 1:3 stoichiometry in the heterotetramer. A higher ratio of PKD1 in the complex may weaken the association of the TOP domains. CONCLUSION The structure of the truncated PKD1-PKD2 complex reveals the molecular mechanism for the assembly of a hetero-oligomeric complex and provides a physical basis for mapping and understanding a large number of disease mutations. Elucidation of the functional mechanism of PKD1 and PKD2 as well as the disease mechanism of the hundreds of ADPKD mutations await further investigations. Our structure serves as a framework for future biophysical, biochemical, cellular, and computational analysis of PKD1-PKD2 and ADPKD. Cryo-EM structure of the truncated human PKD1-PKD2 complex at 3.6-Å resolution. (A) Topological illustration of PKD1 and PKD2. NTD, N-terminal domain; TOP, also known as the polycystin domain; CTD, C-terminal domain (which includes S1 to S6 and the TOP domain). (B) The 1:3 organization of the PKD1-PKD2 complex. PKD2 I, II, and III are the three PKD2 subunits. (C) Unconventional conformation of the S6 segment in PKD1. The sequences between the S5 and S6 segments are flexible and disordered in PKD1. The extracellular segment of the bent S6 resembles PH1. Mutations in two genes, PKD1 and PKD2, account for most cases of autosomal dominant polycystic kidney disease, one of the most common monogenetic disorders. Here we report the 3.6-angstrom cryo–electron microscopy structure of truncated human PKD1-PKD2 complex assembled in a 1:3 ratio. PKD1 contains a voltage-gated ion channel (VGIC) fold that interacts with PKD2 to form the domain-swapped, yet noncanonical, transient receptor potential (TRP) channel architecture. The S6 helix in PKD1 is broken in the middle, with the extracellular half, S6a, resembling pore helix 1 in a typical TRP channel. Three positively charged, cavity-facing residues on S6b may block cation permeation. In addition to the VGIC, a five–transmembrane helix domain and a cytosolic PLAT domain were resolved in PKD1. The PKD1-PKD2 complex structure establishes a framework for dissecting the function and disease mechanisms of the PKD proteins.

中文翻译：

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人 PKD1/PKD2 复合物的结构

一种与肾脏健康有关的复合体 常染色体显性多囊肾病 (ADPKD) 是一种常见的遗传性疾病，可导致肾功能衰竭。蛋白质 PKD1 和 PKD2 的突变与疾病有关，但这些蛋白质在生理学和疾病方面的功能仍不清楚。PKD1 与化学和机械力刺激的感知有关，而 PKD2 被认为是一种钙离子通道。苏等人。显示跨膜区域形成以 1:3 比例组装的 PKD1-PKD2 复合物。他们的高分辨率冷冻电子显微镜结构证实该复合物采用瞬态受体电位通道结构，具有一些独特的特征。将致病突变映射到结构上表明发病机制可能来自复合物的错误折叠或运输，而不是通道活动的破坏。科学，这个问题 p。eaat9819 该结构为进一步研究与多囊肾病相关的复合物提供了框架。引言 PKD1 和 PKD2 两个基因的突变约占常染色体显性多囊肾病 (ADPKD) 病例的 85% 和 10%，ADPKD 是最常见的单基因疾病之一。然而，基因产物 polycystin-1 和 polycystin-2（PC1 和 PC2，也称为 PKD1 和 PKD2）的生理和病理生理功能尚不清楚。PKD1 包含 4303 个残基，可以作为感受化学和机械力刺激的受体，而 PKD2，其同源四聚体结构符合典型的 II 组瞬时受体电位 (TRP) 通道，被假设为内质网 Ca2+ 释放通道并调节细胞内 Ca2+ 浓度。预计这两种蛋白质在肾上皮的初级纤毛上作为异源寡聚体共存，尽管形成这种复合物的分子基础仍然难以捉摸。基本原理为了研究 PKD1 和 PKD2 的组装，我们试图解析 PKD1-PKD2 复合物的结构。在对最佳构建体和表达系统进行广泛筛选后，通过 FLAG 标记的 PKD1 和 Twin-Strep 标记的 PKD2（以下简称为 PKD1 和 PKD2）的共表达获得了同质复合物。通过亲和纯化和尺寸排阻色谱从 40 至 50 升悬浮人胚胎肾 (HEK) 293F 细胞中获得约 100 μg 复合物。使用单粒子冷冻电子显微镜 (cryo-EM) 将复合物的结构确定为 3.6 埃分辨率。结果 PKD1 和 PKD2 在结构中的比例为 1:3。PKD1 由电压门控离子通道 (VGIC) 折叠组成，该折叠与 PKD2 相互作用以完成域交换 TRP 架构。然而，有几个特征将 PKD1 与规范的 TRP 通道区分开来。PKD1 的 S6 段在中间被破坏，细胞外半部分 S6a 类似于典型 VGIC 中的孔螺旋 1 (PH1)。S5 和 S6a 之间的序列在 EM 图中高度灵活且无序。三个带正电荷的残基——Arg4100、Arg4107、和 His4111——突出到假定的离子传导路径中，可能阻碍 Ca2+ 离子的渗透性。因此，电流结构可能代表潜在的非导电状态。在 PKD1 中，包含五个跨膜螺旋 (TM) 和胞质 PLAT（多囊蛋白 1、脂肪氧化酶和 α 毒素）域的离散折叠域位于 VGIC 折叠之前。PKD1 的细胞外 TOP 结构域，经常以 ADPKD 中的突变为目标，偏离预期的对称位置 15°，在细胞外 TOP 环中留下一个缺口。与 PKD2 亚基之间的同型相互作用相比，PKD1 和 PKD2 之间减弱的界面为异四聚体中 1:3 的化学计量提供了线索。复合物中较高比例的 PKD1 可能会削弱 TOP 域的关联。结论截短的PKD1-PKD2复合物的结构揭示了异寡聚复合物组装的分子机制，并为定位和理解大量疾病突变提供了物理基础。PKD1 和 PKD2 的功能机制的阐明以及数百个 ADPKD 突变的疾病机制有待进一步研究。我们的结构可作为未来对 PKD1-PKD2 和 ADPKD 进行生物物理、生物化学、细胞和计算分析的框架。截断的人类 PKD1-PKD2 复合物的 Cryo-EM 结构，分辨率为 3.6 Å。(A) PKD1 和 PKD2 的拓扑图。NTD，N-末端结构域；TOP，又称多囊蛋白域；CTD，C端域（包括S1到S6和TOP域）。(B) PKD1-PKD2 复合体的 1:3 组织。PKD2 I、II、和 III 是三个 PKD2 亚基。(C) PKD1 中 S6 段的非常规构象。S5 和 S6 段之间的序列在 PKD1 中是灵活且无序的。弯曲的 S6 的细胞外段类似于 PH1。PKD1 和 PKD2 两个基因的突变导致了常染色体显性多囊肾病（最常见的单基因疾病之一）的大多数病例。在这里，我们报告了以 1:3 的比例组装的截短的人类 PKD1-PKD2 复合物的 3.6 埃低温电子显微镜结构。PKD1 包含一个电压门控离子通道 (VGIC) 折叠，它与 PKD2 相互作用以形成域交换但非规范的瞬时受体电位 (TRP) 通道结构。PKD1 中的 S6 螺旋在中间断裂，细胞外半部分 S6a 类似于典型 TRP 通道中的孔螺旋 1。三个带正电，S6b 上面向空腔的残基可能会阻止阳离子渗透。除了 VGIC，PKD1 还解析了一个五跨膜螺旋结构域和一个胞质 PLAT 结构域。PKD1-PKD2 复合结构为剖析 PKD 蛋白的功能和疾病机制建立了框架。