Structure of the human PKD1-PKD2 complex

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.