Structure of the polycystic kidney disease TRP channel Polycystin-2 (PC2)


Mutations in either polycystin-1 (PC1 or PKD1) or polycystin-2 (PC2, PKD2 or TRPP1) cause autosomal-dominant polycystic kidney disease (ADPKD) through unknown mechanisms. Here we present the structure of human PC2 in a closed conformation, solved by electron cryomicroscopy at 4.2-Å resolution. The structure reveals a novel polycystin-specific 'tetragonal opening for polycystins' (TOP) domain tightly bound to the top of a classic transient receptor potential (TRP) channel structure. The TOP domain is formed from two extensions to the voltage-sensor-like domain (VSLD); it covers the channel's endoplasmic reticulum lumen or extracellular surface and encloses an upper vestibule, above the pore filter, without blocking the ion-conduction pathway. The TOP-domain fold is conserved among the polycystins, including the homologous channel-like region of PC1, and is the site of a cluster of ADPKD-associated missense variants. Extensive contacts among the TOP-domain subunits, the pore and the VSLD provide ample scope for regulation through physical and chemical stimuli.

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Figure 1: PC2 structure and channel activity.
Figure 2: PC2 TOP-domain structure.
Figure 3: PC2 TOP-domain interactions.
Figure 4: Architecture of the PC2 pore.
Figure 5: Comparison of the PC2 pore with pores from other VGICs.
Figure 6: Locations of ADPKD-associated missense variants in PC2 and PC1.

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The SGC is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, the Canada Foundation for Innovation, Genome Canada, GlaxoSmithKline, Janssen, Lilly Canada, Merck & Co., the Novartis Research Foundation, the Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, EU/EFPIA Innovative Medicines Initiative (IMI) Joint Undertaking (ULTRA-DD 115766) and the Wellcome Trust (092809/Z/10/Z). R.S. is funded by the British Heart Foundation (RG/10/14/28576). The OPIC electron microscopy facility was funded by a Wellcome Trust JIF award (060208/Z/00/Z) and is supported by a WT equipment grant (093305/Z/10/Z). Work in the laboratory of J.T.H. is supported by a Wellcome Trust Core Award (090532/Z/09/Z) and by the European Research Council under the European Union's Horizon 2020 Research and Innovation Programme (649053). We thank the Diamond Light Source for access to the macromolecular crystallography beamlines, and we thank the Diamond Light Source staff for their help with data collection. We acknowledge the use of the University of Oxford Advanced Research Computing (ARC) facility ( and the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311). We thank all members of the SGC Biotech team, including C. Strain-Damerell; K. Kupinska, D. Wang and K. Ellis. We thank all members of the SGC IMP group, including A. Chu, Y.Y. Dong, A. Quigley and S. Bushell. We thank D. Eberhardt for help with the electrophysiology experiments. We are grateful to G. Berridge and O. Borkowska for help with mass spectrometry and to B. Marsden and D. Damerell, J. Bray, J. Crowe and C. Sluman for bioinformatics support. We thank F. von Delft and T. Krojer for assistance with crystallography infrastructure. We thank D. Norris, A. Edwards, S. Tucker and M. Sansom for critical reading of the manuscript.

Author information




M.G., A.C.W.P. and C.A.S. contributed equally to this project. M.G., A.C.W.P., R.S., J.T.H. and E.P.C. designed the project. M.G. optimized and prepared protein samples for X-ray, cryo-EM and electrophysiology data collection, with assistance from A.T.; C.A.S. and A.C.W.P. established the cryo-EM process at the SGC. M.G., C.A.S., A.C.W.P., J.T.H. and E.P.C. collected EM and X-ray data. A.C.W.P. determined the structures, with assistance from M.G., C.A.S., J.T.H. and E.P.C.; L.S., P.M. and S.M. performed pilot studies for protein production and produced baculovirus-infected cells, and R.C. analyzed the mass spectrometry data. Work by L.S., P.M., S.M. and R.C. was supervised by N.A.B.-B., M.G., E.V. and S.E.-A. performed the electrophysiology experiments, under the supervision of R.S.; J.T.H. supervised the cryo-EM work. E.P.C. was responsible for the overall project design, supervision and management. M.G., A.C.W.P., E.V., S.E.-A., R.S., J.T.H. and E.P.C. analyzed the data and prepared the manuscript.

Corresponding authors

Correspondence to Juha T Huiskonen or Elisabeth P Carpenter.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Sequence alignment for PC2 and related orthologs.

The sequence of human PC2P185-D723 (hsPC2; TRPP1) is shown along with the corresponding PC2 sequences from C. elegans (ce) and D. melanogaster (dm). The alignment also includes sequences for human PKD2L1 (TRPP2; aa 65-603), PKD2L2 (TRPP3; aa 1-534), PKD1 (aa 3635-4147) and PKDREJ (aa 1677-2208). Residues conserved in all sequences are boxed and colored dark blue. Secondary structural elements derived from the PC2 EM model (see Fig. 2a and b) are shown above the alignment and colored by domain. Dashed regions represent residues that were not resolved in the EM model. Glycosylation sites in the human PC2 protein are highlighted by green hexagons. A selection of residues that are mutated in ADPKD patients in PC1 or PC2 are shown as colored circles under the alignment with the amino acid change indicated. The color of the circles indicates the predicted impact of the variant on the structure of the protein (colored according to classification in Supplementary Table 1; yellow: minimal predicted impact on the structure; magenta: serious predicted impact on the structure). Uniprot accession codes: hsPC2 (Q13563), cePC2 (Q9U1S7), dmPC2 (Q9VK95), hsPKD2L1 (Q9P0L9), hsPKD2L2 (Q9NZM6), hsPC1 (P98161) and hsPKDREJ (Q9NTG1). The asterisk (*) indicates the location of a large 90 amino acid insertion in the α2b-β0 loop in the fruit fly sequence (dmPC2, residues 328-418) that has been omitted for clarity.

Supplementary Figure 2 Single-particle cryo-EM of human PC2.

(a) Representative raw particles from an original micrograph movie stack after initial frame alignment and summation. (b) Reference-free 2D class averages of detergent solubilised PC2 particles. (c) Initial ‘spinning top’ 3D model generated from a side-on 2D class average used for initial rounds of 3D classification. (d) Resolution estimation based on the gold standard FSC curve from RELION. The FSC curves for both unmasked and masked models calculated using RELION’s automatic post-refinement masking procedure are shown. The overall resolution is calculated to be 4.22 Å based on FSC 0.143. (e) FSC curve of the final 4.2 Å REFMAC-EM refined model versus the map it was refined against (FSCfull, black line). For the purposes of validation, the (semi-randomised) model was refined against a reconstruction generated from half of the particles and then this refined model was compared to an independent reconstruction generated using the remaining particles. The FSC curves are shown for the unbiased model refined against the first of two independent (half) maps (to which it was refined against; FSCwork; red line) or the same refined model versus the second independent half map (to which is was not refined; FSChalf2, green line). (f-g) Local resolution variation throughout the map estimated by RESMAP. Local resolution variation is color-coded from 4-6 Å. (f) Map contoured at low level to indicate the disordered detergent micelle ring covering the TM regions and the partially ordered C-terminal region. (g) Map contoured at higher level showing the PC2 tetramer viewed in the membrane plane (left) and two perpendicular slices through the density at the level of the TOP domain and around the pore. The central helices surrounding the pore and the core elements of the TOP domain exhibit resolutions around 4 Å. The cytosolic N- and C-termini as well as the S2-S3 and S4-S4L loop regions and the extremities of the TOP domain are less well ordered.

Supplementary Figure 3 Quality of EM density map.

EM density maps are shown for (a) the entire monomer, (b) 4 TM helices (S1-S4; cyan) of the VSLD, (c) helices (S4L, S5, PH, S6; blue) in the pore domain, (d) external helices (α1-3; orange) in the TOP domain, (e) central β sheet of TOP domain along with density for individual strands, (f) three glycosylation sites in the TLC motif of the TOP domain (NAG3-5). The EM density map shown corresponds to the final RELION reconstruction filtered at 4 Å and sharpened with a B-factor of –150 Å2. This sharpened map was used also used as input to REFMAC-EM for model refinement in reciprocal space. The map (dark blue mesh) is contoured at 3.5σ. For the glycosylation sites shown in (f), the unsharpened EM map, contoured at 3.5σ is also shown (cyan mesh). Bulky amino acids that aided sequence assignment and register are highlighted. (g) 8.5 Å Anomalous difference Fourier map calculated from the crystallographic data collected from the EMTS-soaked crystals contoured at 3σ. The map (magenta mesh) is overlaid on the ribbon trace of a PC2 monomer with the positions of the nine cysteine residues highlighted. Hg peaks corresponding to all cysteines in the TM helices are observed. (h) Overall view of PC2 tetramer showing the unsharpened 4.2 Å EM density map. Dotted region indicates characteristic C-terminal ‘bulb’ at end of S6B helices. (i) Final refined model with overlaid unsharpened map contoured at low (4.3σ) level. (j) Possible extension of S6B helix has been modelled along with an additional short C-terminal helix. Density for the helical portions is resolved but the connecting regions are not. (k) Density between S6, pointing towards the pore axis, is compatible with a short helix.

Supplementary Figure 4 Single-channel properties of PC2P185–D723.

Representative single-channel currents of PC2P185-D723 in (a) symmetrical 210 mM KCl at +60 mV and (b) in asymmetrical 810 mM (cis) and 210 mM (trans) KCl solutions at +30 mV and (c) -50 mV are shown. An expanded portion of each trace (highlighted in blue) illustrates the rapid gating between multiple sub-conductance levels and the sudden shifting of gating into different preferred sub-conductance states (shown by **). In each trace, the most frequently visited sub-conductance levels are highlighted with green and purple dotted lines and with arrows. The fully open (O) and closed (C) channel levels are indicated with black dashed lines. The all-points histograms shown on the right were obtained from 2 min of consecutive recording and the colored arrows correspond to the predominant sub-conductance levels identified in those traces. (d) Linear regression of the current-voltage relationships for PC2P185-D723 in the same symmetrical KCl (closed circles, n=5 independent experiments) and gradient KCl (open circles, n=6 independent experiments) ionic conditions as in (a-c). Each data point represents the mean ± s.e.m. (e-f) Bar charts compare the probability of PC2P185-D723 gating to the full open state (PoFULL) (e) and to sub-conductance open states (PoSUB) (f) in symmetrical 210 mM KCl solutions (blue) and in KCl gradient (purple). PoFULL is significantly higher at negative holding potential irrespective of whether the channels were in symmetrical (n=5 independent experiments,*P <0.05) or gradient conditions (n=6 independent experiments,*P<0.05). Under all the conditions examined, PC2P185-D723 channel exhibits high levels of activity preferentially dwelling in sub-conductance states (note the different scales on the y-axis in (e) and (f)). Data shown are the mean values ± s.e.m. (g) Example of bilayer recording where control ‘empty’ liposomes were used. (h) Effect of Ca2+: Typical control single-channel recordings in 210 mM KCl at -60 mV are shown (top trace). A reduction in current amplitude is observed after adding 10 mM trans Ca2+ (middle trace) which is reversed after washout of the Ca2+ (bottom trace). (i) Effect of amiloride: A representative control recording in symmetrical 210 mM KCl at -60 mV is shown in the top trace and the effects of adding symmetrical 1 mM amiloride to both cis and trans chambers is shown in the bottom trace. The majority of recordings were of multiple channels and therefore (j) shows bar charts illustrating the reduction in mean current (mean I) that was observed after addition of 10 mM trans Ca2+ (black bar; n=6 independent experiments;*** P < 0.001) or 1 mM symmetrical amiloride (grey bar; n=5 independent experiments;*** P < 0.001) to channels gating in symmetrical 210 mM KCl at +40 mV (controls, white bars). Data shown are the mean values ± s.e.m. (Student’s t-test, *P<0.05; *** P < 0.001).

Supplementary Figure 5 PC2 fold, topology and VSLD structure.

Comparison of the monomer structure for (a) PC2; (b) TRPV1 (PDB: 3J5P) and (c) VGIC CavAb (PDB: 4MVO) viewed in the plane of the membrane (dotted line). Domains are colored according to domain (as Fig. 2a). The N-terminal Ankyrin repeat domain (ARD) of TRPV1 is colored grey. (d) Superposition of monomer structures of PC2 (cyan), TRPV1 (purple) and CavAb (yellow). The structures were superposed with the SSM matching routine in COOT using the S5-S6 pore domain of PC2 tetramer as a reference (to avoid bias from the differently positioned VSLDs). All 3 structures show similar relative orientations of the pore and VSDs. (e) Two perpendicular views of the VSLD (S1-S4). A cluster of conserved residues at the cytosolic end of the four-helix bundle are show in stick form. The conserved lysine residues on S4 (Lys572, Lys575) are in close proximity to a series of buried negatively charged residues. The sidechains of residues that could not be resolved in the EM maps (due to radiation damage/flexibility) have been modelled for illustrative purposes. (f) Comparison of the location of positively charged residues on PC2’s S4 VSLD helix (light blue) with the S4 voltage sensor helix of NavAb / CavAb (yellow, PDB: 4MVO) which contains four arginines (R1-R4). (g) Superposition of the individual VSLD for PC2, TRPV1 and CavAb (colored as in (d)).

Supplementary Figure 6 Sequence conservation of residues in the interface among the TOP domains, the TLC and the S3–S4 extension.

(a) Three views of the PC2 tetramer (looking from top (external face), side (membrane plane) and bottom (cytoplasmic face)). The molecular surface is shown colored by sequence conservation (variable: yellow, highly conserved: dark blue) based on CONSURF analysis of an alignment of 177 PC2 homolog sequences. The external surface displays minimal sequence conservation apart from a small patch on each TLC extension involved in interdomain interaction. In contrast, the cytoplasmic face displays a high degree of conservation around the S6 bundle crossing and within the exposed face of the VSLD four-helix bundle. (b) Detail of the S3b-S4 interface. The VSLD extension is show in ribbon form and colored by sequence conservation. Residues contributing to the conserved interaction site on the TOP β-sheet are labelled. (c) Schematic cartoon ribbon for PC2 monomer colored by conservation viewed in the same orientation as the side view above in (a). The S3b-S4 interaction site with the outer face of the TOP β-sheet is highly conserved. The S3b-S4 interface shown in panel (b) is highlighted. (d) Interaction of the TOP domain with the pore turret region. The pore turret region (ribbons) interacts with the α1 helix and β5 strand of the adjacent TOP domain (molecular surface; regions from the adjacent protomer are indicated by asterisks). Close contacts are made between the S5-PH loop including a direct hydrogen bond between Gln622 and the backbone carbonyl of Phe457 located in β5b. Residues in the turret region and the molecular surface from the adjacent TOP domain are colored by sequence conservation. A significant conserved patch maps to the TOP domain region that interacts with the pore turret. (e) Internal face view of PC2 monomer (180° rotation compared to panel (c)) highlighting the conserved nature of the TLC1 hairpin extension (dotted oval). (f) Perpendicular view corresponding to the approximate orientation of a neighbouring monomer in the tetramer. The surface that the TLC1 hairpin (shown in (e)) interacts with on the other end of the neighbouring TOP domain is indicated with dotted oval and labelled ‘2’. (g) Detail of the TLC1 hairpin extension interaction site.

Supplementary Figure 7 Location of ADPKD-associated missense variants in PC2 and PC1.

(a) PC2 tetramer shown looking onto TOP domain, from the side and a pore domain/VSLD sliced view. Positions of missense variant sites are highlighted by spheres colored according to the predicted impact of a mutation on the channel structure as indicated in Supplementary Table 1 (low – yellow; high – magenta). Variant sites cluster in the TOP domain and around the top of the pore. Fewer variants lie in the VSLD. (b) ADPKD-associated missense variants of PC1 mapped to a model of PC1 channel-like domain derived from the PC2 structure. It is unclear whether PC1 forms a homotetramer or a heterotetramer with PC2 or other polycystins. As with PC2, missense variants in PC1 cluster in the TOP domain. (c) PC2 variant sites mapped on to either a PC2 monomer cartoon representation (left panel) or molecular surface representation. Only a small percentage of the missense variant sites are exposed on the protein surface with the rest buried. The most closely packed cluster maps to the TOP domain TLC extension arm that participates in TOP-TOP interaction (Gly318/Cys331/Tyr345/Thr448).

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Supplementary Figures 1–7, Supplementary Table 1 and Supplementary Note (PDF 2994 kb)

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Grieben, M., Pike, A., Shintre, C. et al. Structure of the polycystic kidney disease TRP channel Polycystin-2 (PC2). Nat Struct Mol Biol 24, 114–122 (2017).

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