McCarthy AE et al. (2019), Full-Length P2X7 Structures Reveal How Palmitoy...

XB-ART-57030

Cell 2019 Oct 17;1793:659-670.e13. doi: 10.1016/j.cell.2019.09.017.

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Full-Length P2X7 Structures Reveal How Palmitoylation Prevents Channel Desensitization.

McCarthy AE , Yoshioka C , Mansoor SE .

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P2X receptors are trimeric, non-selective cation channels activated by extracellular ATP. The P2X7 receptor subtype is a pharmacological target because of involvement in apoptotic, inflammatory, and tumor progression pathways. It is the most structurally and functionally distinct P2X subtype, containing a unique cytoplasmic domain critical for the receptor to initiate apoptosis and not undergo desensitization. However, lack of structural information about the cytoplasmic domain has hindered understanding of the molecular mechanisms underlying these processes. We report cryoelectron microscopy structures of full-length rat P2X7 receptor in apo and ATP-bound states. These structures reveal how one cytoplasmic element, the C-cys anchor, prevents desensitization by anchoring the pore-lining helix to the membrane with palmitoyl groups. They show a second cytoplasmic element with a unique fold, the cytoplasmic ballast, which unexpectedly contains a zinc ion complex and a guanosine nucleotide binding site. Our structures provide first insights into the architecture and function of a P2X receptor cytoplasmic domain.

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Species referenced: Xenopus laevis
Genes referenced: aopep p2rx7 sh3bp4
GO keywords: ligand-gated ion channel activity

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	Figure 1. Overall Architecture of rP2X7 in the Apo State (A–D) The three-dimensional reconstruction of rP2X7 in the apo state, viewed parallel to the membrane (A), perpendicular to the membrane from the extracellular surface (B), from the center of the TM domain toward the cytoplasm (C), and from the intracellular side of the membrane toward the cytoplasm (D). (E) Ribbon representation of rP2X7 receptor corresponding to the apo state reconstruction shown in (A). Each protomer is colored differently. (F) A close-up side view of the cytoplasmic portion of the TM domain, highlighting how the cytoplasmic cap is composed of domain-swapped β strands (β−1, β0, and β15) from each protomer. To improve visual clarity of the cytoplasmic cap, the C-cys anchor and cytoplasmic ballast were removed. (G) Top-down cross-sectional view of the cytoplasmic cap looking toward the cytoplasm. Residues 29–345 and 395–595 of the receptor were removed to improve visual clarity. Each protomer is a different color. (H) To better visualize the spatial interactions between protomers, one protomer is shown in blue, whereas the other two are shown in gray. (I) Ribbon representation of a single subunit of rP2X7 receptor, highlighting P2X receptor domains, including two new domains found only in the P2X7 subtype: the C-cys anchor and the cytoplasmic ballast. The major domains within a protomer are organized by color: extracellular domain in blue, TM domain in green, cytoplasmic cap in cyan, C-cys anchor in purple, and the cytoplasmic ballast in red. The palmitate groups are colored in sand.
	Figure 2. Ion Channel Pore (A–F) Cartoon representation of rP2X7 structures, shown parallel to the membrane as a side view, top down view of the ion channel pore, and ion permeation pathway, respectively, comparing the apo, closed state (A, C, and E) with the ATP-bound, open state (B, D, and F). Binding of agonist induces conformational changes around the binding pocket that result in outward flexing of the extracellular domain’s core β strands, pulling on TM2, causing outward helix expansion to open the pore. For the pore size plots, colors represent different radii: reddish pink, <1.15 Å; green, between 1.15–2.30 Å; purple, >2.30 Å. The N terminus, TM1, and cytoplasmic ballast were removed from (E) and (F) to improve visual clarity.
	Figure 3. Extracellular Orthosteric Ligand-Binding Site (A and B) View of the orthosteric binding pocket, comparing key interactions with ATP in rP2X7 (A) and hP2X3 (B). Two conserved phenylalanine residues (F188 and F293, rP2X7 numbering), proposed to stabilize the ATP-binding pocket, are also similar in both structures (residues not shown). (C–F) Surface representation of the binding pocket for rP2X7 in the open state (C) and apo state (E) compared with hP2X3 in the open state (D) and apo state (F). The apo binding pocket of rP2X7 contains a narrow channel (<11 Å orifice) that shields the pocket from solvent, limiting ligand access. The apo binding pocket of hP2X3 is significantly more solvent exposed (17 Å orifice). The orthosteric binding pocket is at a subunit interface, with protomer A shown in green for open state of rP2X7, forest green for open state of hP2X3, red for the apo state of rP2X7, and red-purple for the apo state of hP2X3. Protomer B is shown in gray, and protomer C is shown in white.
	Figure 4. P2X7 Cytoplasmic Domain; the C-cys Anchor Is Palmitoylated to Modulate Receptor Function by Preventing Desensitization (A) Surface representation of the apo state of rP2X7, highlighting locations of the C-cys anchor and putative phospholipid binding site. (B) Left: the C-cys anchor is a loop containing six cysteine residues and one serine residue that each face toward the plasma membrane. Right: at least four cysteines (C362, C363, C374, and C377) and one serine (S360) on the C-cys anchor are palmitoylated. Palmitoylation of the C-cys anchor is also present in the open state reconstruction but more easily visualized in the higher-resolution apo state map. The higher-resolution apo state reconstruction also reveals that at least one cysteine (C4) in the N terminus is palmitoylated. Because of domain swapping within the context of a trimeric receptor, the N terminus of one protomer is adjacent to the C-cys anchor of an adjacent protomer, grouping together the palmitoylated moieties of different protomers and effectively interlocking them to the membrane. (C and D) Deletion (rP2X7-ΔCcys) of the C-cys anchor (C) or mutation of residues in the C-cys anchor to alanine (rP2X7-CcysMut) to prevent their palmitoylation (D) both result in channels that, when expressed in Xenopus oocytes, nearly completely desensitize during a 30-s application of 100 μM ATP (black bar). Wild-type rP2X7 receptor (rP2X7-WT) does not desensitize. (E) The putative phospholipid binding site, modeled as a PS, is near the middle of the plasma membrane. Each protomer is a different color.
	Figure 5. P2X7 Cytoplasmic Domain; the Cytoplasmic Ballast Contains a Novel Fold (A) Ribbon representation of one subunit of the open state structure of rP2X7 receptor, shown in orthogonal views. Each of the major P2X7 domains is color coded. The cytoplasmic cap, C-cys anchor, and cytoplasmic ballast domains are highlighted. (B) Ribbon representation of the cytoplasmic ballast of rP2X7 receptor, displaying the secondary structure elements, the two zinc ions, and the guanosine nucleotide. (C) Topology diagram of the cytoplasmic ballast, shown in the same orientation as in (B), revealing a novel fold without structural homology in the PDB. Dashed lines denote un-modeled regions. (D and E) Additional ribbon representations of the cytoplasmic ballast of the rP2X7 receptor, shown in two different orthogonal views from (B), highlighting the relative locations of the zinc ions and the guanosine nucleotide within the ballast fold. The models are missing residues S443–R469 in the apo state and residues S443–R471 in the open state.
	Figure 6. The P2X7 Cytoplasmic Ballast Contains a Dinuclear Zinc Ion Complex and a High-Affinity Guanosine Nucleotide Binding Site (A) The cytoplasmic ballast from each protomer lies beneath the TM domain from an adjacent protomer. (B) The entire cytoplasmic domain, viewed perpendicular to the membrane from the cytoplasm. Residues S443–R469 are missing from the apo state structure, visualized here, because no density is present in the map. (C) A bottom-up view focused on the center of the cytoplasmic domain, perpendicular to the membrane from the cytoplasm, reveals how, in the context of a trimer, helices α12 and α13 of each cytoplasmic ballast form a hexagonal hole (diameter, ∼14 Å) through which the α9 helix from each protomer (labeled A, B, and C) emerges. This is referred to as the cytoplasmic plug. The entire cytoplasmic domain assembles as a trimer, in part through the interactions at the interface formed by helices α9, α12, and α13. (D) The cytoplasmic dinuclear Zn2+ ion complex is coordinated by seven cysteine residues in a tetrahedral geometry. (E and F) The cytoplasmic guanosine nucleotide binding site, occupied by a GDP molecule, shown in two views highlighting the density for GDP (E) and key interactions made with conserved P2X7 residues (F). (G) Measurement of 3H-GDP saturation binding to the purified, detergent-solubilized rP2X7 receptor using SPA. The Kd for GDP binding was directly measured as 40 ± 10 nM. Error bars represent the standard deviation of three measurements. (H) Inhibition of 3H-GDP binding to the rP2X7 receptor by guanosine nucleotides yields Ki values of 70 ± 10 nM and 150 ± 15 nM for GDP and GTP, respectively. Non-guanosine diphosphate nucleotides do not displace 3H-GDP and thus do not bind to the cytoplasmic domain of the P2X7 receptor. Error bars represent the standard deviation of three measurements. (I) The guanosine nucleotide binding pocket is exposed at the interface between two cytoplasmic ballasts, accessible as a docking site for protein-protein interactions. Each protomer is a different color.
	Figure 7. Molecular Mechanism of P2X Receptor Gating (A and B) Cartoon models summarizing the gating mechanism for the desensitizing P2X1–6 receptor subtypes (A) compared with the non-desensitizing P2X7 receptor subtype (B).
	Figure S1. The Cryoelectron Microscopy Data Processing Workflow, Using the Apo State Data of Full-Length rP2X7 as an Example, Related to Figure 1 Particles were picked using DoG Picker and visually checked using RELION. Particles were subjected to multiple rounds of 2D classification in RELION to remove false positive picks. For 3D classification in RELION, an ab initio reconstruction was generated in CryoSPARC and used as the reference model. Multiple rounds of 3D refinements and the final map sharpening were carried out using RELION.
	Figure S2. Functional Studies and the Overall Architecture of Full-Length rP2X7 in the Open State, Related to Figure 1 (A) ATP-induced rP2X7 currents (100 μM ATP, black bar) do not undergo desensitization. (B) Co-application of 10 μM antagonist A-438079 (red bar) inhibits the rP2X7 receptor current induced by 100 μM ATP (black bar). (C-F) The three-dimensional reconstruction of full-length rP2X7 in the ATP-bound, open state viewed parallel to the membrane as a side view (C), perpendicular to the membrane from the extracellular surface (D), from the center of the TM domain, toward the cytoplasm (E), and from the intracellular side of the membrane, toward the cytoplasm (F). (G) Ribbon representation of a trimeric receptor structure corresponding to the ATP-bound, open state reconstruction shown in (C). Each protomer is represented by a different color. (H) Ribbon representation of a single subunit of the open state structure, highlighting various purinergic receptor domains, including two new domains, the C-cys anchor and the cytoplasmic ballast. The major domains within a protomer are organized by color: extracellular domain in blue, TM domain in green, cytoplasmic cap in cyan, C-cys anchor in purple, and the cytoplasmic ballast in red. The palmitate groups are colored in sand. (I) A close-up side view of the cytoplasmic portion of the TM domain highlighting the interactions that form the cytoplasmic cap. The cytoplasmic cap is composed of domain-swapped β strands (β-1, β0, and β15) from each protomer. To improve visual clarity of the cytoplasmic cap, the C-cys anchor and cytoplasmic ballast have been removed. (J) Top-down cross-sectional view of the cytoplasmic cap looking toward the cytoplasm. Residues 29-345 and 395-595 of the receptor have been removed to improve visual clarity. Each protomer is a different color.
	Figure S3. Cryoelectron Microscopy Analysis of Full-Length rP2X7 in the Apo and Open States, Related to Figures 1 and S2 (A) Representative electron micrograph of the apo state rP2X7 data. (B) Selected two-dimensional class averages of the electron micrographs of the apo state rP2X7 data. (C, E) The gold-standard Fourier shell correlation curves for the electron microscopy maps of the apo state data (C) and the open state data (E) of rP2X7 are shown in black for the masked map and blue for the unmasked map, and the Fourier shell correlation curves between the atomic model and the final electron microscopy maps are shown in red. (D, F) Angular distribution of particles used for the refinement of the apo state data (D) and the open state data (F).
	Figure S4. Local Resolution Estimates and Representative Densities of the Reconstructions for the Apo-State and Open-State Data of rP2X7, Related to Figures 1, 4, and S2 (A-F) Local resolution estimation of the reconstructions shown as a side view, a top down view from the extracellular surface, and a bottom up view from the cytoplasmic surface, respectively, for the apo state data (A-C) and open state data (D-F) of rP2X7 receptor. (G, H) Representative protein densities from different regions of the apo state (G) and open state (H) maps of rP2X7 receptor. (I, J) Densities for ATP (I) and putative PS phospholipid (J) in the open state map
	Figure S5. The Pore-Lining Surface of rP2X7 for the Open and Apo States, Related to Figure 2 (A) Coronal section of a surface representation of the open state of rP2X7 reveals five vestibules (upper, central, extracellular, intracellular vestibule I, and intracellular vestibule II) are located on the molecular three-fold axis. Beyond the TM gate, two additional sites of constriction exist along the three-fold axis (cytoplasmic cap and cytoplasmic plug). (B) Pore-lining surface along the entire axis of rP2X7 for open and apo states. The color of each sphere represents a different radius from the receptor center, as calculated by the program HOLE: reddish pink < 1.15 Å, green between 1.15 – 2.30 Å, and purple > 2.30 Å. (C) Plot of pore radius as a function of distance along the pore axis (focused around the TM gate) for the apo state versus the open state of rP2X7. The positions of the residues making up the narrowest radius for the TM gate in each conformational state are labeled. The Cα position of residue L353 is set as zero.
	Figure S6. The Dinuclear Zinc Ion Complex, GDP Nucleotide Densities, and Nucleotide Triphosphate Binding in the Cytoplasmic Ballast of rP2X7 Structures, Related to Figures 1, 5, 6, and S2 (A-F) The cytoplasmic ballast of rP2X7 receptor contains density features in the reconstructions that represent the unexpected findings of a zinc ion complex with two Zn2+ ions (Zn1 and Zn2) and a GDP molecule, respectively, for both the apo state (A-C) and open state (D-F) datasets. (A, D) The zinc ion densities are still present when the maps are contoured to a threshold (σ = 25) where the protein density is no longer clearly visible. (B-C, E-F) The residues surrounding the density make interactions consistent with those expected for GDP. Specifically, R546, K583, and R578 make ionic interactions with the phosphates, R574 and the carbonyl of A567 make hydrogen-bonding interactions with the guanine base, and residues L569, Y550, and F591 contribute van der Waal’s interactions to the guanine base. Each of these residues is conserved in human P2X7. The apo state structure is shown in red (B-C) and the open state structure is shown in green (E-F). The densities shown are from the apo and open state maps obtained using a mask that included the TM and cytoplasmic domain, but excluded the extracellular domain (focused refinement on the cytoplasmic domain). (G) Inhibition of 3H-GDP binding to rP2X7 receptor by guanosine nucleotide triphosphates yields Ki values of 150 ± 15 nM and 100 ± 10 nM for GTP and GTP-γ-S, respectively. Non-guanosine nucleotide triphosphates show significantly lower binding affinity – nucleotides UTP and CTP inhibit 3H-GDP binding to rP2X7 receptor with Ki values of 60 ± 10 μM and 130 ± 3 μM, respectively. Nucleotide TTP does not displace 3H-GDP and thus does not bind to the cytoplasmic domain of P2X7 receptor. (H-I) Nucleotide ATP shows a time-dependent displacement of 3H-GDP from the cytoplasmic domain of P2X7 receptor. While no displacement of 3H-GDP occurs initially (H), an ATP-induced displacement of 3H-GDP (Ki value of 22 ± 10 μM) can be measured after several hours (I). A time-dependent change in the displacement of 3H-GDP did not occur with any other nucleotide. Because ATP also binds to the extracellular domain of P2X7 receptor in the orthosteric ATP binding pocket, further studies are needed to determine if the time-dependent displacement of 3H-GDP is due to direct displacement from ATP binding to the cytoplasmic domain or from allosteric modulation, whereby the affinity of 3H-GDP binding to the cytoplasmic domain is decreased after ATP binds to the extracellular, orthosteric ligand binding pocket. Error bars represent the standard deviation of three measurements.
	Figure S7. GDP Detection Using MRM and Functional Assessment of the Cytoplasmic Ballast, Related to Figures 5 and 6 (A-D) Liquid chromatography tandem mass spectrometry analysis on a purified rP2X7 receptor preparation using multiple reaction monitoring (MRM) of fragment ions (m/z 135 and m/z 152) produced from the parent ion (m/z 444) in positive ESI mode confirm the identification of GDP nucleotide. MRM monitoring ion fragment m/z 152 and ion fragment m/z 135, respectively, for buffer and GDP control (A, C) compared to the receptor sample (B, D). The retention time and ratio of the two ions are consistent with GDP nucleotide. No other nucleotides were detected. (E-H) The functional aspects of rP2X7-ΔBallast are nearly identical to rP2X7-WT with respect to kinetics of activation, lack of desensitization, affinity for ATP, and ion selectivity. (E-F) Both rP2X7-WT (E) and rP2X7-ΔBallast (F) show similar current facilitation, and no desensitization, with repeated exposure to 100 μM ATP (1 minute, black bar) at one minute intervals, for three exposures. (G) ATP-activated dose-response curves for rP2X7-WT (black trace) and rP2X7-ΔBallast (red trace), performed in the absence of divalent cations, show similar EC50 values of 42 ± 3 μM and 52 ± 11 μM, respectively. Error bars represent the standard deviation of three measurements. (H) The ATP-induced (100 μM) two electrode voltage clamp currents for rP2X7-WT (black trace) and rP2X7-ΔBallast (red trace) reversed sign at −8 ± 1 mV and −10 ± 3 mV, respectively. Error bars represent the standard deviation of three measurements.
	Graphical Abstract

References [+] :

Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed