Journal: Nature Communications

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1038/s41467-024-52776-7

Figure Lengend Snippet: Cryo-EM maps of the ( A ) ATP-bound P2X1 receptor and ( C ) NF449-bound P2X1 receptor, with individual chains coloured and the cryo-EM ligand density depicted in violet. Enlarged depiction of Mg-ATP and NF449 fitted into the cryo-EM map. Atomic model and structural characteristics of the ( B ) ATP-bound P2X1 receptor and the ( D ) NF449-bound P2X1 receptor colourised to resemble features of a dolphin. The head domain is purple, the lower body is light blue, the dorsal fin is orange, the upper body is blue, the right flipper is red, the left flipper is yellow, and the fluke is green. The dolphin figure was created in BioRender. Bennetts, F. (2023) BioRender.com/r92x211.

Article Snippet: The full-length human P2X1 sequence (WT-P2X1) with C-terminal tag (CT) containing a 3C-cleavage site followed by muGFP (ultra stable GFP ) and an octa-histidine sequence (P2X1-CT) was synthesised from Integrated DNA Technologies (Iowa, USA) (Supplementary Fig. ).

Techniques: Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1038/s41467-024-52776-7

Figure Lengend Snippet: Cartoon representation of the ( A ) ATP-bound P2X1 receptor and ( B ) NF449-bound P2X1 receptor pore region, coloured by monomer. C Graph of the pore radius along the length of the pore region, with the residues responsible for constricting the pore radii highlighted. The ATP-bound P2X1 receptor is depicted in red, while the NF449-bound P2X1 receptor is represented in blue. The pore radius measured 0.6 Å for the ATP-bound receptor and 1.0 Å for the NF449-bound receptor, marked by the dotted line. D Overlay of the transmembrane domains ( E) and a corresponding graph of the pore radius of the ATP-bound P2X1 receptor (red) and the desensitised state ATP-bound hP2X3 receptor (PDB: 5SVL, green). Pore for each P2X3 receptor is displayed by radius with red, white, and blue, with red indicating the area with the tightest constriction point. F Overlay of the transmembrane domains ( G ) and a corresponding graph of the pore radius of the of the NF449-bound P2X1 receptor (blue) and the closed state TNP-ATP-bound hP2X3 receptor (PDB: 5SVQ, yellow). For all pore volumes, the surface volume of the pore radius is highlighted in red, white, and blue, with red indicating the area with the tightest constriction point.

Article Snippet: The full-length human P2X1 sequence (WT-P2X1) with C-terminal tag (CT) containing a 3C-cleavage site followed by muGFP (ultra stable GFP ) and an octa-histidine sequence (P2X1-CT) was synthesised from Integrated DNA Technologies (Iowa, USA) (Supplementary Fig. ).

Techniques:

Journal: Nature Communications

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1038/s41467-024-52776-7

Figure Lengend Snippet: A Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the orthosteric binding site, illustrating movement in secondary structure with arrows. B Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the lower body and transmembrane domain, illustrating movement in secondary structure with arrows.

Article Snippet: The full-length human P2X1 sequence (WT-P2X1) with C-terminal tag (CT) containing a 3C-cleavage site followed by muGFP (ultra stable GFP ) and an octa-histidine sequence (P2X1-CT) was synthesised from Integrated DNA Technologies (Iowa, USA) (Supplementary Fig. ).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1038/s41467-024-52776-7

Figure Lengend Snippet: A Graphic of the ATP binding site, with key interactions illustrated as black dotted lines and nearby residues labelled. B , C Increasing concentrations of the agonist α,β-methylene ATP on HEK293 cells expressing WT-P2X1 or single residue mutants of the P2X1 receptor. The data is normalised to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. A log(agonist) nonlinear regression curve using a four-parameter model is fitted to the data with the top and bottom constrained to 100 and 0, respectively. Data points are represented as mean values with error bars indicating the standard error of the mean (SEM). All P2X1 receptor variants were derived from four independent experiments conducted in triplicate. D Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps represented in white.

Article Snippet: The full-length human P2X1 sequence (WT-P2X1) with C-terminal tag (CT) containing a 3C-cleavage site followed by muGFP (ultra stable GFP ) and an octa-histidine sequence (P2X1-CT) was synthesised from Integrated DNA Technologies (Iowa, USA) (Supplementary Fig. ).

Techniques: Binding Assay, Expressing, Residue, Derivative Assay, Sequencing

Journal: Nature Communications

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1038/s41467-024-52776-7

Figure Lengend Snippet: α,β-methylene ATP potency at wild-type P2X1 and P2X1 mutant receptors

Article Snippet: The full-length human P2X1 sequence (WT-P2X1) with C-terminal tag (CT) containing a 3C-cleavage site followed by muGFP (ultra stable GFP ) and an octa-histidine sequence (P2X1-CT) was synthesised from Integrated DNA Technologies (Iowa, USA) (Supplementary Fig. ).

Techniques: Mutagenesis

Journal: Nature Communications

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1038/s41467-024-52776-7

Figure Lengend Snippet: A Graphic of the NF449 binding site, with key interactions illustrated as black dotted lines and nearby residues labelled. B Competition radioligand binding of a submaximal concentration of [ 3 H]-α,β-methylene ATP (50 nM) with increasing concentrations of NF449 on HEK293 cells expressing WT-P2X1 receptor. A nonlinear regression one site, fit K i curve is fitted to the data. Data points are represented as the mean with the SEM, derived from 3 independent experiments conducted in duplicate. C , D Calcium ion influx using submaximal concentrations of α,β-methylene ATP (0.316–100 µM, 80–95% of max response per mutant) with increasing concentrations of the antagonist NF449 on HEK293 cells expressing WT-P2X1 and single residue mutants of the P2X1 receptor. Data was normalised to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. A log(antagonist) nonlinear regression curve fit using a four-parameter model is fitted to the data with the top and bottom constrained to 100 and 0, respectively. Data points were represented as the mean with the error bars indicating the SEM. P2X1 receptor variants D170A, M214A, E122A, K140A, and S286A were derived from 3 independent experiments and WT-P2X1, R139A, K215A, K136A, E282A, R292A, K136W, R139P, F188L, and L218T were derived from 4 independent experiments, with each experimental condition conducted in triplicate. E Amino acid sequence alignment of NF449 binding site residues from the human P2X1 receptor, aligned with residues from human P2X receptor subtypes. Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps represented in white.

Article Snippet: The full-length human P2X1 sequence (WT-P2X1) with C-terminal tag (CT) containing a 3C-cleavage site followed by muGFP (ultra stable GFP ) and an octa-histidine sequence (P2X1-CT) was synthesised from Integrated DNA Technologies (Iowa, USA) (Supplementary Fig. ).

Techniques: Binding Assay, Concentration Assay, Expressing, Derivative Assay, Mutagenesis, Residue, Sequencing

Journal: Nature Communications

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1038/s41467-024-52776-7

Figure Lengend Snippet: NF449 potency at wild-type P2X1 and P2X1 receptor mutants

Article Snippet: The full-length human P2X1 sequence (WT-P2X1) with C-terminal tag (CT) containing a 3C-cleavage site followed by muGFP (ultra stable GFP ) and an octa-histidine sequence (P2X1-CT) was synthesised from Integrated DNA Technologies (Iowa, USA) (Supplementary Fig. ).

Techniques:

Journal: Nature Communications

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1038/s41467-024-52776-7

Figure Lengend Snippet: A The ATP Mg 2+ ion binding site modelled into the cryo-EM map of the ATP-bound P2X1 receptor, with surrounding residues, ligands, and waters. B The possible central metal ion binding site cryo-EM density of the ATP-bound P2X1 receptor, with surrounding residues. C Amino acid sequence alignment of D97, E122 and D170 from the human P2X1 receptor (hP2X1: P51575), aligned with the corresponding residue from human P2X receptor subtypes (hP2X2: Q9UBL9, hP2X3: P56373, hP2X4: Q99571, hP2X5: Q93086, hP2X6: O15547, hP2X7: Q99572). Residue colourisation based on properties: Hydrophobic: blue, positive: red, negative: magenta, polar: green, special cases: orange, gaps: white. D Validation metrics for the magnesium ion positioned adjacent to ATP, colour-coded as follows: green: acceptable, yellow: borderline, and red: poor. No metal ion could be conclusively identified at the potential central metal ion site. E Increasing concentrations of the agonist α,β-methylene ATP on HEK293 cells expressing WT-P2X1 or P2X1 receptor mutants. The data is normalised to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. A log(agonist) nonlinear regression curve using a four-parameter model is fitted to the data with the top and bottom constrained to 100 and 0, respectively. Data points represent the mean, with error bars indicating the SEM, from four independent experiments conducted in triplicate. F Saturation radioligand binding on HEK293 cells expressing WT-P2X1 receptor or P2X1 receptor mutants. A nonlinear regression one site, fit K i curve is fitted to the data. Data points represent the mean, with error bars indicating the SEM. P2X1 receptor variants WT-P2X1 were derived from 5 independent experiments, D170A and E122A from 3, and D97A from 2, with each condition performed in triplicate. Values that exceeded 300 nM were classified as not defined. G Calculated EC 50 and K i values from experiments conducted on WT-P2X1 or P2X1 receptor mutants. Statistically significant differences compared to WT-P2X1 were calculated using a one-way ANOVA with a Dunnett’s multiple comparison test. P values: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

Article Snippet: The full-length human P2X1 sequence (WT-P2X1) with C-terminal tag (CT) containing a 3C-cleavage site followed by muGFP (ultra stable GFP ) and an octa-histidine sequence (P2X1-CT) was synthesised from Integrated DNA Technologies (Iowa, USA) (Supplementary Fig. ).

Techniques: Binding Assay, Cryo-EM Sample Prep, Sequencing, Residue, Expressing, Derivative Assay, Comparison

Journal: Nature Communications

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1038/s41467-024-52776-7

Figure Lengend Snippet: Cryo-EM maps of the ( A ) ATP-bound P2X1 receptor and ( C ) NF449-bound P2X1 receptor, with individual chains coloured and the cryo-EM ligand density depicted in violet. Enlarged depiction of Mg-ATP and NF449 fitted into the cryo-EM map. Atomic model and structural characteristics of the ( B ) ATP-bound P2X1 receptor and the ( D ) NF449-bound P2X1 receptor colourised to resemble features of a dolphin. The head domain is purple, the lower body is light blue, the dorsal fin is orange, the upper body is blue, the right flipper is red, the left flipper is yellow, and the fluke is green. The dolphin figure was created in BioRender. Bennetts, F. (2023) BioRender.com/r92x211.

Article Snippet: The full-length human P2X1 sequence (WT-P2X1) with C-terminal tag (CT) containing a 3C-cleavage site followed by muGFP (ultra stable GFP ) and an octa-histidine sequence (P2X1-CT) was synthesised from Integrated DNA Technologies (Iowa, USA) (Supplementary Fig. ).

Techniques: Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1038/s41467-024-52776-7

Figure Lengend Snippet: Cartoon representation of the ( A ) ATP-bound P2X1 receptor and ( B ) NF449-bound P2X1 receptor pore region, coloured by monomer. C Graph of the pore radius along the length of the pore region, with the residues responsible for constricting the pore radii highlighted. The ATP-bound P2X1 receptor is depicted in red, while the NF449-bound P2X1 receptor is represented in blue. The pore radius measured 0.6 Å for the ATP-bound receptor and 1.0 Å for the NF449-bound receptor, marked by the dotted line. D Overlay of the transmembrane domains ( E) and a corresponding graph of the pore radius of the ATP-bound P2X1 receptor (red) and the desensitised state ATP-bound hP2X3 receptor (PDB: 5SVL, green). Pore for each P2X3 receptor is displayed by radius with red, white, and blue, with red indicating the area with the tightest constriction point. F Overlay of the transmembrane domains ( G ) and a corresponding graph of the pore radius of the of the NF449-bound P2X1 receptor (blue) and the closed state TNP-ATP-bound hP2X3 receptor (PDB: 5SVQ, yellow). For all pore volumes, the surface volume of the pore radius is highlighted in red, white, and blue, with red indicating the area with the tightest constriction point.

Article Snippet: The full-length human P2X1 sequence (WT-P2X1) with C-terminal tag (CT) containing a 3C-cleavage site followed by muGFP (ultra stable GFP ) and an octa-histidine sequence (P2X1-CT) was synthesised from Integrated DNA Technologies (Iowa, USA) (Supplementary Fig. ).

Techniques:

Journal: Nature Communications

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1038/s41467-024-52776-7

Figure Lengend Snippet: A Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the orthosteric binding site, illustrating movement in secondary structure with arrows. B Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the lower body and transmembrane domain, illustrating movement in secondary structure with arrows.

Article Snippet: The full-length human P2X1 sequence (WT-P2X1) with C-terminal tag (CT) containing a 3C-cleavage site followed by muGFP (ultra stable GFP ) and an octa-histidine sequence (P2X1-CT) was synthesised from Integrated DNA Technologies (Iowa, USA) (Supplementary Fig. ).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1038/s41467-024-52776-7

Figure Lengend Snippet: A Graphic of the ATP binding site, with key interactions illustrated as black dotted lines and nearby residues labelled. B , C Increasing concentrations of the agonist α,β-methylene ATP on HEK293 cells expressing WT-P2X1 or single residue mutants of the P2X1 receptor. The data is normalised to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. A log(agonist) nonlinear regression curve using a four-parameter model is fitted to the data with the top and bottom constrained to 100 and 0, respectively. Data points are represented as mean values with error bars indicating the standard error of the mean (SEM). All P2X1 receptor variants were derived from four independent experiments conducted in triplicate. D Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps represented in white.

Article Snippet: The full-length human P2X1 sequence (WT-P2X1) with C-terminal tag (CT) containing a 3C-cleavage site followed by muGFP (ultra stable GFP ) and an octa-histidine sequence (P2X1-CT) was synthesised from Integrated DNA Technologies (Iowa, USA) (Supplementary Fig. ).

Techniques: Binding Assay, Expressing, Residue, Derivative Assay, Sequencing

Journal: Nature Communications

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1038/s41467-024-52776-7

Figure Lengend Snippet: α,β-methylene ATP potency at wild-type P2X1 and P2X1 mutant receptors

Article Snippet: The full-length human P2X1 sequence (WT-P2X1) with C-terminal tag (CT) containing a 3C-cleavage site followed by muGFP (ultra stable GFP ) and an octa-histidine sequence (P2X1-CT) was synthesised from Integrated DNA Technologies (Iowa, USA) (Supplementary Fig. ).

Techniques: Mutagenesis

Journal: Nature Communications

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1038/s41467-024-52776-7

Figure Lengend Snippet: A Graphic of the NF449 binding site, with key interactions illustrated as black dotted lines and nearby residues labelled. B Competition radioligand binding of a submaximal concentration of [ 3 H]-α,β-methylene ATP (50 nM) with increasing concentrations of NF449 on HEK293 cells expressing WT-P2X1 receptor. A nonlinear regression one site, fit K i curve is fitted to the data. Data points are represented as the mean with the SEM, derived from 3 independent experiments conducted in duplicate. C , D Calcium ion influx using submaximal concentrations of α,β-methylene ATP (0.316–100 µM, 80–95% of max response per mutant) with increasing concentrations of the antagonist NF449 on HEK293 cells expressing WT-P2X1 and single residue mutants of the P2X1 receptor. Data was normalised to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. A log(antagonist) nonlinear regression curve fit using a four-parameter model is fitted to the data with the top and bottom constrained to 100 and 0, respectively. Data points were represented as the mean with the error bars indicating the SEM. P2X1 receptor variants D170A, M214A, E122A, K140A, and S286A were derived from 3 independent experiments and WT-P2X1, R139A, K215A, K136A, E282A, R292A, K136W, R139P, F188L, and L218T were derived from 4 independent experiments, with each experimental condition conducted in triplicate. E Amino acid sequence alignment of NF449 binding site residues from the human P2X1 receptor, aligned with residues from human P2X receptor subtypes. Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps represented in white.

Article Snippet: The full-length human P2X1 sequence (WT-P2X1) with C-terminal tag (CT) containing a 3C-cleavage site followed by muGFP (ultra stable GFP ) and an octa-histidine sequence (P2X1-CT) was synthesised from Integrated DNA Technologies (Iowa, USA) (Supplementary Fig. ).

Techniques: Binding Assay, Concentration Assay, Expressing, Derivative Assay, Mutagenesis, Residue, Sequencing

Journal: Nature Communications

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1038/s41467-024-52776-7

Figure Lengend Snippet: NF449 potency at wild-type P2X1 and P2X1 receptor mutants

Article Snippet: The full-length human P2X1 sequence (WT-P2X1) with C-terminal tag (CT) containing a 3C-cleavage site followed by muGFP (ultra stable GFP ) and an octa-histidine sequence (P2X1-CT) was synthesised from Integrated DNA Technologies (Iowa, USA) (Supplementary Fig. ).

Techniques:

Journal: Nature Communications

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1038/s41467-024-52776-7

Figure Lengend Snippet: A The ATP Mg 2+ ion binding site modelled into the cryo-EM map of the ATP-bound P2X1 receptor, with surrounding residues, ligands, and waters. B The possible central metal ion binding site cryo-EM density of the ATP-bound P2X1 receptor, with surrounding residues. C Amino acid sequence alignment of D97, E122 and D170 from the human P2X1 receptor (hP2X1: P51575), aligned with the corresponding residue from human P2X receptor subtypes (hP2X2: Q9UBL9, hP2X3: P56373, hP2X4: Q99571, hP2X5: Q93086, hP2X6: O15547, hP2X7: Q99572). Residue colourisation based on properties: Hydrophobic: blue, positive: red, negative: magenta, polar: green, special cases: orange, gaps: white. D Validation metrics for the magnesium ion positioned adjacent to ATP, colour-coded as follows: green: acceptable, yellow: borderline, and red: poor. No metal ion could be conclusively identified at the potential central metal ion site. E Increasing concentrations of the agonist α,β-methylene ATP on HEK293 cells expressing WT-P2X1 or P2X1 receptor mutants. The data is normalised to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. A log(agonist) nonlinear regression curve using a four-parameter model is fitted to the data with the top and bottom constrained to 100 and 0, respectively. Data points represent the mean, with error bars indicating the SEM, from four independent experiments conducted in triplicate. F Saturation radioligand binding on HEK293 cells expressing WT-P2X1 receptor or P2X1 receptor mutants. A nonlinear regression one site, fit K i curve is fitted to the data. Data points represent the mean, with error bars indicating the SEM. P2X1 receptor variants WT-P2X1 were derived from 5 independent experiments, D170A and E122A from 3, and D97A from 2, with each condition performed in triplicate. Values that exceeded 300 nM were classified as not defined. G Calculated EC 50 and K i values from experiments conducted on WT-P2X1 or P2X1 receptor mutants. Statistically significant differences compared to WT-P2X1 were calculated using a one-way ANOVA with a Dunnett’s multiple comparison test. P values: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

Article Snippet: The full-length human P2X1 sequence (WT-P2X1) with C-terminal tag (CT) containing a 3C-cleavage site followed by muGFP (ultra stable GFP ) and an octa-histidine sequence (P2X1-CT) was synthesised from Integrated DNA Technologies (Iowa, USA) (Supplementary Fig. ).

Techniques: Binding Assay, Cryo-EM Sample Prep, Sequencing, Residue, Expressing, Derivative Assay, Comparison

Journal: bioRxiv

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1101/2024.04.04.588192

Figure Lengend Snippet: A) Cryo-EM map of the ATP-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of Mg-ATP and nearby water molecules was fitted into the cryo-EM map. B) Cryo-EM map of the NF449-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of NF449 was fitted into the cryo-EM map. C) Structural characteristics of the monomer from the ATP-bound P2X1 receptor and the NF449-bound P2X1 receptor colourised to resemble features of a dolphin. The head domain is colored purple, the lower body is light blue, the dorsal fin is orange, the upper body is blue, the right flipper is red, the left flipper is yellow, and the fluke is green.

Article Snippet: To measure ATP bound to detergent purified P2X1 receptor a Kinase-Glo® Luminescent Kinase Assay (ProMega, USA) was used.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1101/2024.04.04.588192

Figure Lengend Snippet: Cartoon representation of the A) ATP-bound P2X1 receptor and B) NF449-bound P2X1 receptor coloured by monomer. The surface volume of the pore radius is highlighted in red, white, and blue, with red indicating the area with the tightest constriction point. C) Graph of the pore radius along the length of the pore region with the residues responsible for constricting the pore radii highlighted. The ATP-bound P2X1 receptor is depicted in blue, while the NF449-bound P2X1 receptor is represented in red. The pore radius measured 0.6 Å for the ATP-bound receptor and 1.0 Å for the NF449-bound receptor, indicated by the dotted line. D) Transmembrane domains of the ATP-bound P2X1 receptor (red) are overlaid with those of the ATP-bound hP2X3 receptor (5SVL, green). Additionally, transmembrane domains of the NF449-bound P2X1 receptor (blue) are overlaid with those of the TNP-ATP-bound hP2X3 receptor (5SVQ, yellow).

Article Snippet: To measure ATP bound to detergent purified P2X1 receptor a Kinase-Glo® Luminescent Kinase Assay (ProMega, USA) was used.

Techniques:

Journal: bioRxiv

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1101/2024.04.04.588192

Figure Lengend Snippet: A) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the orthosteric binding site, illustrating movement in secondary structure with arrows. B) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the lower body and transmembrane domain, illustrating movement in secondary structure with arrows.

Article Snippet: To measure ATP bound to detergent purified P2X1 receptor a Kinase-Glo® Luminescent Kinase Assay (ProMega, USA) was used.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1101/2024.04.04.588192

Figure Lengend Snippet:

Article Snippet: To measure ATP bound to detergent purified P2X1 receptor a Kinase-Glo® Luminescent Kinase Assay (ProMega, USA) was used.

Techniques: Derivative Assay, Expressing, Residue

Journal: bioRxiv

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1101/2024.04.04.588192

Figure Lengend Snippet: A) Depiction of the ATP binding site where the key interactions are illustrated as black dotted lines, accompanied by labeling of nearby residues. B, C) Increasing concentrations of the agonist α,β-methylene ATP on HEK293 cells expressing WT-P2X1 or single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(agonist) nonlinear regression curve using a four-parameter model, with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 4 replicates. The left graph contains mutants designed for the ATP-bound P2X1 receptor and the right graph contains mutants designed for the NF449-bound P2X1 receptor. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.

Article Snippet: To measure ATP bound to detergent purified P2X1 receptor a Kinase-Glo® Luminescent Kinase Assay (ProMega, USA) was used.

Techniques: Binding Assay, Labeling, Expressing, Residue, Sequencing

Journal: bioRxiv

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1101/2024.04.04.588192

Figure Lengend Snippet: A) Depiction of the NF449 binding site where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. B, C) Submaximal concentrations of α,β-methylene ATP (0.316 - 100 µM, 80-95% of max per mutant) with increasing concentrations of the antagonist NF449 on HEK293 cells expressing WT-P2X1 and single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(antagonist) nonlinear regression curve fit (four parameters) with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 3 - 4 replicates. The left graph contains data for single alanine mutants, and the right graph contains data for P2X1 to P2X7 single residue mutants. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.

Article Snippet: To measure ATP bound to detergent purified P2X1 receptor a Kinase-Glo® Luminescent Kinase Assay (ProMega, USA) was used.

Techniques: Binding Assay, Mutagenesis, Expressing, Residue, Sequencing

Journal: bioRxiv

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1101/2024.04.04.588192

Figure Lengend Snippet: Magnified image of the ATP binding site at the P2X1, hP2X3 (5SVL), zfP2X4 (4DW1), and rP2X7 (6U9W) receptor structures where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. RMSD of ATP in the P2X1 receptor compared to bound ATP in the hP2X3, zfP2X4, and rP2X7 receptors: 0.70, 0.93, and 1.3, respectively.

Article Snippet: To measure ATP bound to detergent purified P2X1 receptor a Kinase-Glo® Luminescent Kinase Assay (ProMega, USA) was used.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1101/2024.04.04.588192

Figure Lengend Snippet: Overlay of the NF449 binding site from the P2X1 receptor structure (blue) compared to A-317491 from the hP2X3 receptor (5SVR, violet), PPNDS from the pdP2X4 receptor (8JV8, green), and TNP-ATP from the ckP2X7 receptor (5XW6, yellow). The 2D chemical structures for each of these ligands are depicted.

Article Snippet: To measure ATP bound to detergent purified P2X1 receptor a Kinase-Glo® Luminescent Kinase Assay (ProMega, USA) was used.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1101/2024.04.04.588192

Figure Lengend Snippet: A) Cryo-EM map of the ATP-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of Mg-ATP and nearby water molecules was fitted into the cryo-EM map. B) Cryo-EM map of the NF449-bound P2X1 receptor, with chains coloured individually and the cryo-EM ligand density depicted in violet. An enlarged image of NF449 was fitted into the cryo-EM map. C) Structural characteristics of the monomer from the ATP-bound P2X1 receptor and the NF449-bound P2X1 receptor colourised to resemble features of a dolphin. The head domain is colored purple, the lower body is light blue, the dorsal fin is orange, the upper body is blue, the right flipper is red, the left flipper is yellow, and the fluke is green.

Article Snippet: The sample was loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva, USA), and fractions containing the trimeric P2X1 receptor were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80⁰C.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1101/2024.04.04.588192

Figure Lengend Snippet: Cartoon representation of the A) ATP-bound P2X1 receptor and B) NF449-bound P2X1 receptor coloured by monomer. The surface volume of the pore radius is highlighted in red, white, and blue, with red indicating the area with the tightest constriction point. C) Graph of the pore radius along the length of the pore region with the residues responsible for constricting the pore radii highlighted. The ATP-bound P2X1 receptor is depicted in blue, while the NF449-bound P2X1 receptor is represented in red. The pore radius measured 0.6 Å for the ATP-bound receptor and 1.0 Å for the NF449-bound receptor, indicated by the dotted line. D) Transmembrane domains of the ATP-bound P2X1 receptor (red) are overlaid with those of the ATP-bound hP2X3 receptor (5SVL, green). Additionally, transmembrane domains of the NF449-bound P2X1 receptor (blue) are overlaid with those of the TNP-ATP-bound hP2X3 receptor (5SVQ, yellow).

Article Snippet: The sample was loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva, USA), and fractions containing the trimeric P2X1 receptor were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80⁰C.

Techniques:

Journal: bioRxiv

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1101/2024.04.04.588192

Figure Lengend Snippet: A) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the orthosteric binding site, illustrating movement in secondary structure with arrows. B) Overlay of the ATP-bound P2X1 receptor (red) and NF449-bound P2X1 receptor (blue) at the lower body and transmembrane domain, illustrating movement in secondary structure with arrows.

Article Snippet: The sample was loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva, USA), and fractions containing the trimeric P2X1 receptor were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80⁰C.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1101/2024.04.04.588192

Figure Lengend Snippet:

Article Snippet: The sample was loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva, USA), and fractions containing the trimeric P2X1 receptor were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80⁰C.

Techniques: Derivative Assay, Expressing, Residue

Journal: bioRxiv

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1101/2024.04.04.588192

Figure Lengend Snippet: A) Depiction of the ATP binding site where the key interactions are illustrated as black dotted lines, accompanied by labeling of nearby residues. B, C) Increasing concentrations of the agonist α,β-methylene ATP on HEK293 cells expressing WT-P2X1 or single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(agonist) nonlinear regression curve using a four-parameter model, with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 4 replicates. The left graph contains mutants designed for the ATP-bound P2X1 receptor and the right graph contains mutants designed for the NF449-bound P2X1 receptor. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.

Article Snippet: The sample was loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva, USA), and fractions containing the trimeric P2X1 receptor were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80⁰C.

Techniques: Binding Assay, Labeling, Expressing, Residue, Sequencing

Journal: bioRxiv

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1101/2024.04.04.588192

Figure Lengend Snippet: A) Depiction of the NF449 binding site where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. B, C) Submaximal concentrations of α,β-methylene ATP (0.316 - 100 µM, 80-95% of max per mutant) with increasing concentrations of the antagonist NF449 on HEK293 cells expressing WT-P2X1 and single residue mutants of the P2X1 receptor. The data is normalized to 10 µM ionomycin and subsequently adjusted so that the highest value corresponds to 100 and the lowest value to 0. It is then fitted to a log(antagonist) nonlinear regression curve fit (four parameters) with the top and bottom constrained to 100 and 0, respectively. Results are presented as mean ± SEM, with n = 3 - 4 replicates. The left graph contains data for single alanine mutants, and the right graph contains data for P2X1 to P2X7 single residue mutants. D) Amino acid sequence alignment of key residues from the human P2X1 receptor (hP2X1:P51575), aligned with corresponding residues from other human P2X receptor subtypes (hP2X2:Q9UBL9, hP2X3:P56373, hP2X4:Q99571, hP2X5:Q93086, hP2X6:O15547, hP2X7:Q99572). Residue categorisation based on properties: Hydrophobic (A, I, L, M, F, W, V, Y) in blue, positive charge (K, R, H) in red, negative charge (E, D) in magenta, polar (N, Q, S, T) in green. Special cases (C, G, P) in orange. Gaps are represented in white.

Article Snippet: The sample was loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva, USA), and fractions containing the trimeric P2X1 receptor were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80⁰C.

Techniques: Binding Assay, Mutagenesis, Expressing, Residue, Sequencing

Journal: bioRxiv

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1101/2024.04.04.588192

Figure Lengend Snippet: Magnified image of the ATP binding site at the P2X1, hP2X3 (5SVL), zfP2X4 (4DW1), and rP2X7 (6U9W) receptor structures where the key interactions are illustrated as black dotted lines, accompanied by labelling of nearby residues. RMSD of ATP in the P2X1 receptor compared to bound ATP in the hP2X3, zfP2X4, and rP2X7 receptors: 0.70, 0.93, and 1.3, respectively.

Article Snippet: The sample was loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva, USA), and fractions containing the trimeric P2X1 receptor were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80⁰C.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Structural insights into the human P2X1 receptor and ligand interactions

doi: 10.1101/2024.04.04.588192

Figure Lengend Snippet: Overlay of the NF449 binding site from the P2X1 receptor structure (blue) compared to A-317491 from the hP2X3 receptor (5SVR, violet), PPNDS from the pdP2X4 receptor (8JV8, green), and TNP-ATP from the ckP2X7 receptor (5XW6, yellow). The 2D chemical structures for each of these ligands are depicted.

Article Snippet: The sample was loaded onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva, USA), and fractions containing the trimeric P2X1 receptor were pooled, concentrated, flash-frozen in liquid nitrogen, and stored at −80⁰C.

Techniques: Binding Assay