X-ray structures define human P2X3 receptor gating cycle and antagonist action

Abstract

P2X receptors are trimeric, non-selective cation channels activated by ATP that have important roles in the cardiovascular, neuronal and immune systems. Despite their central function in human physiology and although they are potential targets of therapeutic agents, there are no structures of human P2X receptors. The mechanisms of receptor desensitization and ion permeation, principles of antagonism, and complete structures of the pore-forming transmembrane domains of these receptors remain unclear. Here we report X-ray crystal structures of the human P2X3 receptor in apo/resting, agonist-bound/open-pore, agonist-bound/closed-pore/desensitized and antagonist-bound/closed states. The open state structure harbours an intracellular motif we term the ‘cytoplasmic cap’, which stabilizes the open state of the ion channel pore and creates lateral, phospholipid-lined cytoplasmic fenestrations for water and ion egress. The competitive antagonists TNP-ATP and A-317491 stabilize the apo/resting state and reveal the interactions responsible for competitive inhibition. These structures illuminate the conformational rearrangements that underlie P2X receptor gating and provide a foundation for the development of new pharmacological agents.

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Figure 1: Architecture and pore structure for major conformational states of the gating cycle of hP2X3.
Figure 2: Apo to open state transition.
Figure 3: Open to desensitized state transition.
Figure 4: Orthosteric ligand-binding site.
Figure 5: Extracellular and cytoplasmic fenestrations.
Figure 6: The gating cycle.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates for the structure have been deposited in the Protein Data Bank under the accession codes 5SVJ, 5SVK, 5SVL, 5SVM, 5SVP, 5SVQ, 5SVR, 5SVS, and 5SVT.

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Acknowledgements

We thank M. Hattori for initial construct screening, L. Vaskalis for figures, H. Owen for manuscript preparation and Gouaux laboratory members for discussions. We acknowledge the Berkeley Center for Structural Biology at the Advanced Light source for assistance with data collection at beamline 5.0.2 and the Northeastern Collaborative Access Team at the Advanced Photon Source for assistance with data collection at beamline 24-ID-C. The simulations were supported by the National Institutes of General Medical Sciences (U54-GM087519 and P41-GM104601 to E.T.) and computationally through XSEDE (TG-MCA06N060 to E.T.). E.G. is an investigator with the Howard Hughes Medical Institute. This research was supported by the National Institute of General Medical Sciences (5F32GM108391 to S.E.M and R01GM100400 to E.G.).

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Authors

Contributions

S.E.M. and E.G. designed the project. S.E.M. performed the biochemical and functional analyses. S.E.M. and W.O. carried out the protein purification and crystallization. S.E.M., W.L., and W.O. performed the crystallography and model building. M.S. and E.T. performed the molecular dynamics simulations. All authors wrote and edited the manuscript.

Corresponding author

Correspondence to Eric Gouaux.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks P. Biggin, R. Murrell-Lagnado and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Functional studies of hP2X3-MFC and hP2X3-MFCslow.

a, Measurement of [3H-ATP] saturation binding to purified, detergent-solubilized hP2X3-MFC using SPA. For each point in the plot, the error bars indicate the standard error of the mean (SEM) for triplicate samples. The calculated Kd for ATP binding was 2.8 ± 0.1 nM and represents the average of two separate experiments. b, ATP-induced currents for hP2X3-WT and hP2X3-MFC both show rapid desensitization kinetics with τ = 523 ± 198 ms and 429 ± 43 ms, respectively. These values represent an average of three measurements with error values indicating s.e.m. Actual rate constants are likely to be faster as the perfusion rate of our TEVC system is ~1,000 ms. c, Measurement of [3H-ATP] saturation binding to purified, detergent solubilized hP2X3-MFCslow using SPA. The calculated Kd for ATP binding was 3.3 ± 0.3 nM. d, ATP-induced currents for hP2X3-MFCslow show delayed desensitization kinetics with τ = 42,581 ± 2,194 ms. e, f, Co-application of 2 μM TNP-ATP (e) or 2 μM A-317491 (f) inhibits the current induced by 1 μM ATP for hP2X3-WT, hP2X3-MFC and hP2X3-MFCslow. g, Inhibition of 3H-ATP binding to hP2X3-MFCslow by unlabelled TNP-ATP yields a Ki of 94 ± 12 nM. Inhibition of 3H-ATP binding to hP2X3-MFC by unlabelled TNP-ATP yields a Ki of 118 ± 1 nM (data not shown). For each point in the plot, the error bars indicate the s.e.m. for triplicate samples. The reported Ki values represent the average of two separate experiments. h, i, Co-application of 2 μΜ TNP-ATP (h) or 2 μΜ A-317491 (i) blocks the residual current remaining after prolonged application of 1 μM ATP to hP2X3-MFCslow receptors.

Extended Data Figure 2 Naming of purinergic receptor domains and comparison of hP2X3 structures to previously published zfP2X4 structures.

a, Ribbon representation of one subunit of the open state structure of hP2X3 receptor shown in orthogonal views. The new cytoplasmic cap domain is termed the ‘tail fin’. b, Cartoon representation of the open state hP2X3 structure aligned to the open state zfP2X4 structure (construct name ΔP2X4-C) shown parallel to the membrane as a side view and as viewed perpendicular to the membrane from the extracellular side. The transmembrane domains for the hP2X3 structure are longer and more complete than for the zfP2X4 structure. c, Cartoon representation of the apo state hP2X3 structure aligned to the apo state zfP2X4 structure (construct name ΔP2X4-B) shown parallel to the membrane as a side view and viewed perpendicular to the membrane from the extracellular side. d, Sequence alignment of the N terminus (top alignment) and C terminus (bottom alignment) of hP2X3 compared to zfP2X4. Starting and ending residues of the hP2X3 construct compared to the ΔP2X4-C construct are indicated with red arrows. The hP2X3 crystallization construct has more residues at both termini than the ΔP2X4-C crystallization construct.

Extended Data Figure 3 The pore-lining surface of hP2X3 for the open, apo and desensitized states.

a, A coronal section of a surface representation of the open state of hP2X3 reveals that four vestibules (upper, central, extracellular and intracellular) are located on the molecular three-fold axis. b, Pore-lining surfaces along the entire axis of hP2X3 for open, apo and desensitized states. The colour of each sphere represents a different radius from the receptor centre, as calculated by the program HOLE: red <1.15 Å, green 1.15–2.30 Å, purple >2.30 Å. c, Plot of pore radius as a function of distance along the pore axis for the open state versus the apo state versus the desensitized state. The positions of the residues making up the narrowest radius in each conformational state are labelled. The Cα position of I341 is set as zero. I323 defines the first constriction site of the gate (extracellular boundary of the gate), whereas T330 defines the second constriction site (cytoplasmic boundary of the gate). These residues are at the equivalent positions that define the boundaries of the gate in the apo state structure of zfP2X4, but are leucine and alanine residues, respectively, in zfP2X4. A single residue, V334, defines the constriction site of the desensitized state. Residue T330 defines the narrowest region of the pore in the open state.

Extended Data Figure 4 The overall structure and ion channel pore of antagonist-bound/closed states.

a, b, Cartoon representation of the competitive antagonist-bound/closed state structures, TNP-ATP in cyan (a) and A-317491 in blue (b), shown parallel to the membrane as a side view. c, An overall alignment of a single protomer in the apo state (red-purple), TNP-ATP-bound state (cyan) and A-317491-bound state (blue). d, Plot of pore radius as a function of distance along the pore axis for apo state versus TNP-ATP-bound state versus A-317491-bound state. The positions of the residues making up the narrowest radius in each conformational state are labelled. The Cα position of I341 is set as zero. e, f, Pore-lining surfaces along the entire axis of the receptor and a focus on the transmembrane domain with TM2 pore-lining residues shown as sticks for the TNP-ATP-bound state (e) and the A-317491-bound state (f). The colour of each sphere represents a different radius from the receptor centre, as calculated by the program HOLE: red <1.15 Å, green 1.15–2.30 Å, purple >2.30 Å.

Extended Data Figure 5 High-affinity P2X3 agonist 2-methylthio-ATP can be soaked into the desensitized state crystals.

a, Inhibition of 3H-ATP binding to hP2X3-MFC by unlabelled 2-methylthio-ATP yields a Ki of 1.9 ± 0.1 nM. For each point in the plot, the error bars indicate the s.e.m. for triplicate samples. The reported Ki represents the average of two separate experiments. b, Electron density for ATP in the desensitized state. The FoFc map is contoured at 1.0σ. c, Desensitized state crystals that have been soaked with 2-methylthio-ATP have a density in the binding pocket, which matches the shape of 2-methylthio-ATP, accounting for the methyl-thio moiety. The FoFc map is contoured at 1.0σ. d, An anomalous difference Fourier map (contoured at 3.0σ) has anomalous signal that overlaps with the sulfur moiety of 2-methylthio-ATP as well as the phosphate groups. These crystals of hP2X3-MFC successfully ligand-exchanged ATP for agonist 2-methylthio-ATP in the binding pocket but were destroyed when soaked with antagonist TNP-ATP, providing evidence that the structure represents an agonist-bound, closed or desensitized state.

Extended Data Figure 6 Resetting from desensitized to apo state of hP2X3.

a, b, Structures of hP2X3 in the desensitized state (a) and apo state (b) shown parallel to the membrane. There are marked changes between the two states in the extracellular domain and the transmembrane domain. c, d, Top-down view comparing the pore of the desensitized state (c) to the pore of the apo state (d) highlighting how, although both pores are closed, the residues that define the gates are different. e, Relative differences in the pore between desensitized and apo states after aligning the upper body domain of the trimer reveal that a significant clockwise conformational change at both the extracellular and cytoplasmic surfaces of the transmembrane domain must occur for the receptor pore to reset back to the apo state. f, Alignment of TM2 in desensitized versus apo state shows that both helices have the same helical pitch, suggesting that the 310-helix that existed in the open state is a transient structural feature. The inset shows the view along the axis of the TM2 helix, observed from the cytoplasmic surface. We speculate that the structural resetting of the receptor from the desensitized state to the apo state is likely to occur after ligand dissociation.

Extended Data Figure 7 Orthosteric ligand-binding site and ligand densities.

a, b, View of the orthosteric binding pocket for the ATP-bound open state structure of hP2X3. ATP binds at an interface between two protomers, with protomer A shown in green and protomer B shown in grey. The 2FoFc density for ATP is shown at 2.5σ. c, d, View of the orthosteric binding pocket for the TNP-ATP-bound closed state structure of hP2X3 with protomer A shown in cyan and protomer B shown in grey. The 2 FoFc density for TNP-ATP is shown at 1.5σ. e, f, View of the orthosteric binding pocket for the A-317491-bound closed state structure of hP2X3 with protomer A shown in blue and protomer B shown in grey. The 2FoFc density for A-317491 is shown at 0.8σ. g, Close-up comparison of the relative orientation of ATP (shown as translucent) versus TNP-ATP in the binding pocket highlights how the phosphate moiety and the orientation of the ribose group are both inverted between the two molecules. h, The apo state structure (shown in figure) as well as both antagonist-bound structures have a Mg2+ ion present in the head domain of hP2X3, coordinated by the side chains of E109 and D158 as well as the carbonyl oxygen of E156. The 2FoFc density for the Mg2+ ion is shown at 1.5σ.

Extended Data Figure 8 Anomalous signal from Mn2+ ion proves Mg2+ ion is present in the head domain of the apo state.

a, Anomalous difference map of apo structure with crystals grown in MnCl2 have an anomalous signal from a Mn2+ ion in the head domain (anomalous difference Fourier map shown in green contoured at 5.5σ). This anomalous signal from Mn2+ overlaps with the 2FoFc density shown in Extended Data Fig. 7h, proving this density is a Mg2+ ion. b, The Mn2+ ion in the head domain is coordinated by the side chains of E109 and D158 and the carbonyl oxygen of E156. c, The presence of a Mg2+ ion does not change the affinity of ATP for hP2X3-MFCslow, as assessed by SPA binding, suggesting that Mg2+ does not compete with ATP for the binding pocket or impair the ability of ATP to bind to the receptor. For each point in the plot, the error bars indicate the s.e.m. for triplicate measurements. The reported Kd values represent the mean of two separate experiments.

Extended Data Table 1 Data collection and refinement statistics
Extended Data Table 2 Anomalous data collection statistics

Supplementary information

Demonstration of the overall structural conformational changes that occur during the gating cycle of hP2X3 receptor

This video demonstrates the overall structural conformational changes that occur during the gating cycle of hP2X3 receptor, highlighting the receptor's transition from the apo state to the open state to the desensitized state before resetting back to the apo state. Each protomer subunit is shown in a different colour. (MOV 9747 kb)

Structural conformational changes that occur in the receptor's pore during the gating cycle of hP2X3, highlighting the transition of the pore from the apo state to the open state to the desensitized state before resetting back to the apo state.

This video demonstrates the structural conformational changes that occur in the receptor's pore during the gating cycle of hP2X3, shown perpendicular to the membrane from the extracellular surface. It highlights the transition of the pore from the apo state to the open state to the desensitized state before resetting back to the apo state. Each protomer subunit is shown in a different colour. (MOV 10426 kb)

Structural conformational changes that occur in the receptor’s pore during the gating cycle of hP2X3, highlighting the residues that define the narrowest region across the pore in each conformational state: I323 in the apo state (red-purple), T330 in the open state (green) and V334 in the desensitized state (yellow).

This video shows the structural conformational changes that occur in the receptor’s pore during the gating cycle of hP2X3, shown perpendicular to the membrane from the extracellular surface, highlighting the residues that define the narrowest region across the pore in each conformational state: I323 in the apo state (red-purple), T330 in the open state (green), and V334 in the desensitized state (yellow). The movie steps through the transition of the pore from the apo state to the open state to the desensitized state before resetting back to the apo state. (MOV 9739 kb)

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Mansoor, S., Lü, W., Oosterheert, W. et al. X-ray structures define human P2X3 receptor gating cycle and antagonist action. Nature 538, 66–71 (2016). https://doi.org/10.1038/nature19367

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