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.).