High-affinity agonism at the P2X7 receptor is mediated by three residues outside the orthosteric pocket

Abstract

P2X receptors are trimeric ATP-gated ion channels that activate diverse signaling cascades. Due to its role in apoptotic pathways, selective activation of P2X₇ is a potential experimental tool and therapeutic approach in cancer biology. However, mechanisms of high-affinity P2X₇ activation have not been defined. We report high-resolution cryo-EM structures of wild-type rat P2X₇ bound to the high-affinity agonist BzATP as well as significantly improved apo receptor structures in the presence and absence of sodium. Apo structures define molecular details of pore architecture and reveal how a partially hydrated Na⁺ ion interacts with the conductance pathway in the closed state. Structural, electrophysiological, and direct binding data of BzATP reveal that three residues just outside the orthosteric ATP-binding site are responsible for its high-affinity agonism. This work provides insights into high-affinity agonism for any P2X receptor and lays the groundwork for development of subtype-specific agonists applicable to cancer therapeutics.

Fig. 1. Global architecture and Na⁺ ion coordination in the apo closed state pore of rP2X₇.

A Ribbon representation of the updated apo closed state structure of rP2X₇ at 2.5 Å, with one protomer colored by domain name, the second colored in pale green, and the third colored in gray. B View of the Na⁺ ion (purple sphere) found directly above the pore’s constriction site (created by TM2 from each protomer) in the apo closed state with its electron density map shown in blue mesh. The Na⁺ ion is partially hydrated, coordinated in octahedral geometry by three symmetry-related water molecules (red spheres) and the sidechain hydroxyl of S342 from each protomer (~2.3 Å for all interactions). Directly below the gate and within the pore, three additional symmetry-related water molecules are poised to re-hydrate the ion once the channel opens (3 Å hydrogen bond distance to the sidechain hydroxyl of S342). C The same view as in panel (B) of apo rP2X₇ purified in the absence of sodium, highlighting the lack of density for an ion above the gate. This result confirms the identity of the density shown in panel (B) as a partially hydrated Na⁺ ion. D Zoomed out view of panel (B) highlighting the topology of the TM2 helix, stabilized by interactions with three different sets of symmetry-related ordered water molecules, one set within the pore and above the gate and the other two sets below the gate and within the transmembrane domain. TM2 starts as a standard alpha helix (green ribbon), then transitions to a short 3₁₀-helix (cyan ribbon, residues 340–344) just above the constriction gate and shortly after, transitions back to a standard alpha helix (green ribbon) just below the constriction gate. Transitions to and from the 3₁₀-helix are mediated by ordered water molecules within the transmembrane domain that induce kinks in the helical backbone of TM2. Interactions with the protein are in gold dashed lines and interactions for Na⁺ coordination are in black dashed lines. All sidechains in TM2 are hidden except for the sidechains of S339 and S342.

Fig. 2. Activation and binding kinetics of ATP and BzATP to rP2X₇.

A TEVC recording of rP2X₇ in response to 100 μM ATP, highlighting fast activation after repeated exposure to ATP, no significant desensitization, and brisk deactivation. The reported deactivation lifetime (τ), shown in reddish-orange, is the time it takes for current to return to 63% of baseline after removal of ATP. B Dose response curves from TEVC experiments measured the activation of full-length wild-type rP2X₇ by ATP (orange) and BzATP (blue), resulting in EC₅₀ values of 34 ± 9 μM and 1.2 ± 0.2 μM, respectively. Data points and error bars represent the mean and standard deviation of normalized current across triplicate experiments, respectively. C, D Representative BLI sensorgrams for a dilution series of ATP (C, shades of orange) and BzATP (D, shades of blue) binding to biotinylated rP2X₇ immobilized on streptavidin (SA) biosensors. Kinetic data were globally fit using a Langmuir 1:1 model to determine the equilibrium dissociation constant (K_D) of ATP to rP2X₇ as K_D = 540 ± 230 nM (C) and the equilibrium dissociation constant of BzATP to rP2X₇ as K_D = 7.4 ± 2.7 nM (D), representing the mean and standard deviation across triplicate experiments, respectively. For kinetic analysis, a 90 s association time and a 200 s dissociation time were used for both analytes. However, to optimize visualization, only 120 s of the dissociation time is shown in the figure.

Fig. 3. Structural comparison of rP2X₇ bound to BzATP vs ATP.

A Ribbon representation of the BzATP-bound open state structure of rP2X₇ solved to 2.8 Å colored by protomer (blue, light-blue, and gray) highlighting the orthosteric ligand-binding site (red box). B–E Magnified view of the red box from (A) at different scales. The ATP-bound rP2X₇ structure (PDB code: 6U9W) and the BzATP-bound rP2X₇ structure were aligned in ChimeraX. B Comparison of positions and rotamers of residues in the orthosteric ligand-binding site (ligands hidden), which are conserved across all P2XRs, that directly interact with ATP for the ATP-bound rP2X₇ structure (orange and light gray) and the BzATP-bound rP2X₇ structure (blue and gray). In the register of rP2X₇, the conserved orthosteric residues that interact with ATP across all P2XRs are K64, K66, T189, K193, N292, R294, K311. C Comparison of ATP-bound (orange) and BzATP-bound (blue) structures, focusing on the residues just outside the canonical orthosteric ligand-binding site that make direct interactions with BzATP. With the ATP molecule hidden but the BzATP molecule shown, the panel highlights the 5 Å movement and 90^o rotation of a loop in the head domain (residues 123–128) containing residue R125 that occurs when BzATP is bound. In the ATP-bound model, R125 is stubbed at the Cβ carbon. First comparative view (D, E) and second comparative view (F, G), rotated 90 degrees from (C–E), of the rP2X₇ orthosteric ligand-binding site with ATP bound (orange and light gray) vs. BzATP bound (blue and gray). D View of the orthosteric ligand-binding site of ATP-bound rP2X₇ highlighting the rotamer of I214 and the position of R125, which lacks interactions with ATP. E View of the orthosteric ligand-binding site of BzATP-bound rP2X₇ highlighting the rotamer of I214 and the position of R125, which is now well-defined. F View of the orthosteric ligand-binding site of ATP-bound rP2X₇ highlighting the rotamer of Q143, facing away from ATP, unable to make direct interactions to ATP. G View of the orthosteric ligand-binding site of BzATP-bound rP2X₇ highlighting the position and rotamer of Q143, now participating in several hydrogen bonding interactions with BzATP and residue R125.

Fig. 4. Apparent affinities (EC₅₀) of rP2X₇ activation by ATP and BzATP.

A Dose-response curves (EC₅₀) for wild-type rP2X₇ activated by ATP and BzATP as well as the three single mutant receptors (R125A, Q143A, and I214A) activated by BzATP. These traces highlight the impact of specific residues just outside the canonical orthosteric ligand-binding site on the apparent affinity of the receptor for BzATP. No single residue mutation is able to fully abrogate the higher apparent affinity of BzATP relative to ATP. B Dose-response curves (EC₅₀) for the three double mutant receptors (R125A/Q143A, R125A/I214A, and Q143A/I214A) activated by ATP and BzATP. While each of the double mutant receptors demonstrated a decreased apparent affinity for ATP relative to wild-type rP2X₇ receptor (Table 1), in each case, the corresponding apparent affinity for BzATP always remained significantly higher. C Dose-response curves (EC₅₀) for wild-type and triple mutant (R125A/Q143A/I214A) rP2X₇ activated by ATP and BzATP highlighting that mutation of all three residues just outside the canonical orthosteric ligand-binding site completely eliminates the high-affinity agonism of BzATP relative to ATP. Note that while the wild-type receptor has a ~28-fold higher apparent affinity for BzATP than ATP, the apparent affinity of the triple mutant receptor (R125A/Q143A/I214A) is essentially identical for ATP and BzATP. The apparent affinity (EC₅₀) values for all wild-type and mutant receptors are included in Table 1. Data points and error bars represent the mean and standard deviation of normalized current across triplicate experiments, respectively.

Fig. 5. Deactivation rates of rP2X₇ following activation by ATP and BzATP.

A–F Representative TEVC traces of wild-type and mutant rP2X₇ highlighting the differences in the rates of receptor deactivation, represented by τ, following activation by ATP or BzATP. A, C, E Representative TEVC traces of wild-type and mutant (R125A and R125A/I214A) rP2X₇ highlighting a fast rate of receptor deactivation following activation by ATP and a slow rate of receptor deactivation following activation by BzATP. B, D, F Representative TEVC traces of mutant (Q143A, R125A/Q143A, and R125A/Q143A/I214A) rP2X₇ highlighting a fast rate of receptor deactivation following activation by both ATP and BzATP. A TEVC traces of wild-type rP2X₇ highlighting the different deactivation kinetics following activation by ATP compared to BzATP. B TEVC traces highlighting the impact of the only single mutation (Q143A) to affect the rate of receptor deactivation. C TEVC traces highlighting that mutation of the residue (R125) with the biggest structural change upon BzATP binding has little effect on the rate of receptor deactivation. D TEVC traces highlighting that double mutations which include Q143A display a fast rate of receptor deactivation following activation by BzATP. E TEVC traces highlighting that the double mutation (R125A/I214A), which does not include Q143A, retains a slow rate of receptor deactivation following activation by BzATP. F TEVC traces highlighting that the triple mutant receptor (R125A/Q143A/I214A) has the same fast rate of receptor deactivation following activation by ATP and BzATP. The complete list of deactivation times (τ) for all wild-type and mutant constructs are included in Table 1, with data points and error bars representing the mean and standard deviation of normalized currents across triplicate experiments, respectively.

Fig. 6. Efficacy of rP2X₇ activation by ATP and BzATP.

(Left) Normalized current from TEVC experiments of wild-type rP2X₇ in response to two concentrations of ATP and BzATP, highlighting the increased normalized responses from BzATP as compared to ATP. This is consistent with BzATP acting as a full agonist and ATP as a partial agonist. (Right) Normalized current from TEVC experiments of triple mutant (R125A/Q143A/I214A) in response to two concentrations of ATP and BzATP, highlighting the virtually identical normalized responses to ATP and BzATP. The triple mutation renders BzATP and ATP equivalent agonists in terms of efficacy. Each replicate was normalized to the current from a 100 μM application of ATP so multiple oocytes could be tested. Data points and error bars represent the mean and standard deviation of normalized current across triplicate experiments, respectively.

Fig. 7. Structural basis for high-affinity agonism at the P2X₇ receptor.

Schematic representation of the more compacted orthosteric pocket when BzATP is bound to rP2X₇ compared to when ATP is bound. A View of ATP bound in the orthosteric rP2X₇ ligand-binding site highlighting the positions of residues R125, Q143, and I214. Residue R125 is stubbed at the Cβ due to a lack of density in the map. B View of ATP bound in the orthosteric rP2X₇ ligand-binding site highlighting a more open pocket. C View of BzATP bound in the orthosteric rP2X₇ ligand-binding site highlighting a more compact pocket. D View of BzATP bound in the orthosteric rP2X₇ ligand-binding site highlighting the positions of residues R125, Q143, and I214. B, C Because of its key interactions with residues R125, Q143, and I214 in rP2X₇, BzATP has a faster association rate (k_a) and a slower dissociation rate (k_d), contributing to its higher binding affinity (equilibrium dissociation constant, K_D) compared to ATP.