Figures and data
Cryo-EM structures of PPNDS-bound and PPADS-bound pdP2X7.
The trimeric structures of PPNDS-bound (A) and PPADS-bound (B) pdP2X7, viewed parallel to the membrane. The PPNDS and PPADS molecules are shown as spheres. Each subunit of the trimers is colored blue, yellow, and red. The EM density maps contoured at 4.5 σ and 3.5 σ for PPNDS and PPADS are shown as gray mesh. The structural formulas of PPNDS and PPADS are also shown.
Binding site for PPNDS
(A, B) Overall structure (A) and close-up view of the PPNDS binding site (B) in the PPNDS-bound pdP2X7 structure. PPNDS molecules are shown by stick models. Water molecules are depicted as red spheres. Dotted black lines indicate hydrogen bonding.
Binding site for PPADS
(A, B) Overall structure (C) and close-up view of the PPADS binding site (D) in the PPADS-bound pdP2X7 structure. PPADS molecules are shown by stick models. Water molecules are depicted as red spheres. Dotted black lines indicate hydrogen bonding.
ATP binding site and sequence comparison
(A) Overall structure and close-up view of the ATP-bound rat P2X7 structure (PDB ID: 6U9W). The cytoplasmic domain is not shown. Dotted black lines indicate hydrogen bonding. (B) Sequence alignment of Ailuropoda melanoleuca P2X7 (pdP2X7) (Accession number: XP_002913164.3), Rattus norvegicus (rP2X7) (Accession number: Q64663.1) and Homo sapiens P2X receptors (P2X1: P51575.1, P2X2: Q9UBL9.1, P2X3: P56373.2, P2X4: Q99571.2, P2X5: Q93086.4, P2X6: O15547.2, and P2X7: Q99572.4). Orange, green and red circles indicate the residues involved in ATP, PPADS and PPNDS recognition.
Structural comparison and inhibition mechanism
(A) Superposition of the ATP-bound rP2X7 structure (red, PDB ID: 6U9W) and the PPNDS-bound pdP2X7 structure (green, this study) onto the apo rP2X7 structure (gray, PDB ID: 6U9V). Close-up views of the head, left flipper, and lower body domains and the intracellular view of the TM domain are shown in each box. Arrows indicate the conformational changes from the apo to ATP-bound states (red) and from the apo to the PPNDS-bound states (green). (B) A cartoon model of the PPNDS/PPADS-dependent inhibition and ATP-dependent activation mechanisms.
Structure-based mutational analysis
(A) Superimposition of the PPNDS-bound and PPADS-bound structures in this study onto the predicted human P2X1 structure (AlphaFold). Each subunit of the PPNDS-bound and PPADS-bound structures is shown in blue, yellow, and red, while the predicted human P2X1 structure is shown in gray. The PPNDS and PPADS molecules and the residues surrounding PPNDS and PPADS that are different between pdP2X7 and hP2X1 are shown as sticks. (B) Effects of PPNDS (10 µM) on ATP (1 mM)-evoked currents of pdP2X7 and its mutants (mean ± SD, n = 5). (C) Effects of PPNDS (1 µM) on ATP (1 µM)-evoked currents of hP2X1 and its mutants (mean ± SD, n = 5-10). (D) Effects of PPNDS (10 µM) on ATP (1 µM)-evoked currents of hP2X3 and its mutants (mean ± SD, n = 5, one-way ANOVA post hoc test, **: p <0.01, ****: p <0.0001 vs. WT.)
Effects of PPNDS and PPADS on P2X receptors by patch clamp recording
(A-D) Representative current traces from patch clamp recordings of P2X receptors. Effects of 10 µM PPNDS (blue) on the 1 mM ATP-evoked (orange) current of pdP2X7 and its mutants (A). Effects of 100 µM PPADS (blue) on the 1 mM ATP-evoked (orange) current of pdP2X7 (B). Effects of 1 µM PPNDS (blue) on the 1 µM ATP-evoked (orange) current of hP2X1 (C). Effects of 10 µM PPNDS (blue) on the 1 µM ATP-evoked (orange) current of hP2X3 (D). (E-F) Effects of PPNDS (10 µM) (E) and PPADS (100 µM) (F) on ATP (1 mM)-evoked currents of pdP2X7 (mean ± SD, n = 5). The graph for PPNDS was taken from Fig. 6B. The inhibition ratio is defined by normalizing the peak current amplitude from the coapplication of PPNDS/PPADS and ATP to the peak current amplitude from the ATP application prior to the coapplication of PPNDS/PPADS and ATP.
Cryo-EM analysis of PPNDS-bound pdP2X7
(A) The gold -standard Fourier shell correlation curves for the PPNDS -bound data. (B) Angular particle distribution. The heat map of particle projections in each viewing angle. (C) The side view, a top-down view from the extracellular surface and a bottom-up view from the intracellular surface colored by local resolution.
Cryo-EM data process for PPNDS-bound pdP2X7
Before the C1 symmetry, all steps were performed in Relion 3.1. Further 3D classification using Cryosparc v4.2.1 by non -uniform refinement of this final set of particles resulted in a cryo-EM map at 3.34 Å resolution.
Cryo-EM analysis of PPADS-bound pdP2X7
(A) The gold -standard Fourier shell correlation curves for the PPADS -bound data. (B) Angular particle distribution. The heat map of particle projections in each viewing angle. (C) The side view, a top-down view from the extracellular surface and a bottom-up view from the intracellular surface colored by local resolution.
Cryo-EM data process for PPADS-bound pdP2X7
Before the C1 symmetry, all steps were performed in Relion 3.1. Further 3D classification using Cryosparc v4.2.1 by non -uniform refinement of this final set of particles resulted in a cryo-EM map at 3.60 Å resolution.
Dolphin model
(A) The P2X7 protomer in cartoon representation. Each structural feature is colored according to the dolphin model. (B, C) Superposition of the PPNDS -bound structure (green) onto the PPADS -bound structure (gray). Each protomer is shown in cartoon representations, and PPNDS and PPADS are shown in stick representations (B). The intracellular view of thetransmembrane domain and the residues at the constriction regio are shown in stick representations (C).
EM density maps for the PPNDS and PPADS binding sites
(A, B) Close -up views of the PPNDS (A) and PPADS (B) binding sites. Dotted lines represent hydrogen bonds. The EM density maps for the residues involved in the PPNDS and PPADS interactions are shown and contoured at 5.0 σ and 4.0 σ, respectively.
MD simulations of the PPNDS-bound pdP2X7 structure
(A, B) The plots of the root mean square deviations (RMSD) of Cα atoms (A) and the RMSD values of atoms in PPNDS (B).
