Identification of a carbohydrate recognition motif of purinergic receptors
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eLife assessment
Purines are native molecules that affect processes in the immune system, among others. The manuscript describes a valuable investigation of the mode of binding of purines, especially their carbohydrate moiety, to human receptors in cell culture and by computer-based modelling. Solid evidence is presented about the way purines interact with and activate two receptors.
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Abstract
As a major class of biomolecules, carbohydrates play indispensable roles in various biological processes. However, it remains largely unknown how carbohydrates directly modulate important drug targets, such as G-protein coupled receptors (GPCRs). Here, we employed P2Y purinoceptor 14 (P2Y14), a drug target for inflammation and immune responses, to uncover the sugar nucleotide activation of GPCRs. Integrating molecular dynamics simulation with functional study, we identified the uridine diphosphate (UDP)-sugar-binding site on P2Y14, and revealed that a UDP-glucose might activate the receptor by bridging the transmembrane (TM) helices 2 and 7. Between TM2 and TM7 of P2Y14, a conserved salt bridging chain (K 2.60 -D 2.64 -K 7.35 -E 7.36 [KDKE]) was identified to distinguish different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-glucuronic acid, and UDP- N -acetylglucosamine. We identified the KDKE chain as a conserved functional motif of sugar binding for both P2Y14 and P2Y purinoceptor 12 (P2Y12), and then designed three sugar nucleotides as agonists of P2Y12. These results not only expand our understanding for activation of purinergic receptors but also provide insights for the carbohydrate drug development for GPCRs.
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Author Response
Reviewer #1 (Public Review):
Zhao et al. investigated the molecular nature of the binding site for carbohydrates within the UDP-sugars known to activate the P2Y14 receptor. In order to do so, they built a molecular model of the hP2Y14, docked the corresponding agonists, and performed MD simulation on the resulting complexes. The modeling was used to identify the key molecular interactions with a cluster of charged residues in the extracellular side of the TM region of the receptor, which they show are conserved within the P2Y receptors. The binding site of the UDP region was, not surprisingly, overlapping with the analogous ADP binding site experimentally observed for the P2Y12 receptor, and consequently, the region that recognizes the sugars could be anticipated. Nevertheless, the detailed modeling and simulation …
Author Response
Reviewer #1 (Public Review):
Zhao et al. investigated the molecular nature of the binding site for carbohydrates within the UDP-sugars known to activate the P2Y14 receptor. In order to do so, they built a molecular model of the hP2Y14, docked the corresponding agonists, and performed MD simulation on the resulting complexes. The modeling was used to identify the key molecular interactions with a cluster of charged residues in the extracellular side of the TM region of the receptor, which they show are conserved within the P2Y receptors. The binding site of the UDP region was, not surprisingly, overlapping with the analogous ADP binding site experimentally observed for the P2Y12 receptor, and consequently, the region that recognizes the sugars could be anticipated. Nevertheless, the detailed modeling and simulation work shows the consistency of this hypothesis and provides a quantification of the particular interactions involved, pinpointing specifically the residues candidate to be involved in the recognition of sugars.
It follows the characterization, by functional assays, of the effect of single-point mutations of these residues in the efficacy of the different UDP-sugars. Here the results show a tendency to correlate with the molecular models, however some of the data has very low statistical significance and consequently the interpretation and conclusions extracted from this data should be taken with caution. This pertains to the particular role of the identified residues in the binding of the different sugars, which in some cases should be taken as a suggestion rather than a proof, though the general conclusion of the identification of the binding region for the sugar, its conservation among P2Y receptors and the role of some specific residues in sugar recognition seems convincing and the data are conveniently presented.
Finally, the design of ADP-sugars that activate the P2Y12 receptor, based on the transferability of the observations with the UDP-sugars for the P2Y14 receptor, is a first indication that such a recognition is possible and should happen in an analogous binding region. However, the low potencies exhibited by the ADP-sugars, in the micromolar range, are too far from the ADP agonist and the relevance of this mechanism remains to be proved. The difference between P2Y12 and P2Y14, with the last one showing much higher potencies for UDP-sugar derivatives than P2Y12 for the corresponding ADP-sugars, remains an interesting question not explored in this manuscript.
Thanks for your valuable comments. We have revised the interpretation of the data that has relatively low statistical significance in the manuscript. The conclusions extracted from this data have also been modified as suggestions. In this work, to investigate whether sugar nucleotides can also activate human P2Y12, we tested three ADP-sugars for human P2Y12. Discovery of highly potent P2Y12 agonists requires screening of a large number of compounds. It is possible there are the other ADP-sugars, which are highly potent P2Y12 agonists. It is technically challenging to synthesize ADP-sugars. Currently, we can only obtain ADP-Glc, ADP-GlcA and ADP-Man. Once the other ADP-sugars are available for us, we will test them and try to discover highly potent agonists in the future work. The highly potent agonists will be useful chemical tools to unveil the relevance mechanism of P2Y12. To explore the nature of binding site of the P2Y12 and P2Y14, we performed more experiments of mutagenesis study and added relevant data in the revised manuscript.
Reviewer #2 (Public Review):
The manuscript employs multiple approaches, including molecular docking, molecular dynamic simulations, and functional experiments to uncover a distinct uridine diphosphate-sugar-binding site on P2Y14 - a key drug target for inflammation and immune responses. Overall, the manuscript is clearly written, and the experimental techniques are well-documented. However, it may benefit from further analysis, particularly in terms of validating the binding pose.
Thanks for your comments. We used MMPBSA to analyze the ligand-binding energy for each receptor residue using MD trajectories. To further characterize the ligand-binding pose, we calculated the percentage of occurrence of hydrogen binding between the ligand and the carbohydrate-binding site (K277, E278, R253 and K77). We also calculated the ligand RMSF and ligand RMSD to show the stability of the ligand-binding pose and the simulation convergence. These data have been included in the revised manuscript.
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eLife assessment
Purines are native molecules that affect processes in the immune system, among others. The manuscript describes a valuable investigation of the mode of binding of purines, especially their carbohydrate moiety, to human receptors in cell culture and by computer-based modelling. Solid evidence is presented about the way purines interact with and activate two receptors.
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Reviewer #1 (Public Review):
Zhao et al. investigated the molecular nature of the binding site for carbohydrates within the UDP-sugars known to activate the P2Y14 receptor. In order to do so, they built a molecular model of the hP2Y14, docked the corresponding agonists, and performed MD simulation on the resulting complexes. The modeling was used to identify the key molecular interactions with a cluster of charged residues in the extracellular side of the TM region of the receptor, which they show are conserved within the P2Y receptors. The binding site of the UDP region was, not surprisingly, overlapping with the analogous ADP binding site experimentally observed for the P2Y12 receptor, and consequently, the region that recognizes the sugars could be anticipated. Nevertheless, the detailed modeling and simulation work shows the …
Reviewer #1 (Public Review):
Zhao et al. investigated the molecular nature of the binding site for carbohydrates within the UDP-sugars known to activate the P2Y14 receptor. In order to do so, they built a molecular model of the hP2Y14, docked the corresponding agonists, and performed MD simulation on the resulting complexes. The modeling was used to identify the key molecular interactions with a cluster of charged residues in the extracellular side of the TM region of the receptor, which they show are conserved within the P2Y receptors. The binding site of the UDP region was, not surprisingly, overlapping with the analogous ADP binding site experimentally observed for the P2Y12 receptor, and consequently, the region that recognizes the sugars could be anticipated. Nevertheless, the detailed modeling and simulation work shows the consistency of this hypothesis and provides a quantification of the particular interactions involved, pinpointing specifically the residues candidate to be involved in the recognition of sugars.
It follows the characterization, by functional assays, of the effect of single-point mutations of these residues in the efficacy of the different UDP-sugars. Here the results show a tendency to correlate with the molecular models, however some of the data has very low statistical significance and consequently the interpretation and conclusions extracted from this data should be taken with caution. This pertains to the particular role of the identified residues in the binding of the different sugars, which in some cases should be taken as a suggestion rather than a proof, though the general conclusion of the identification of the binding region for the sugar, its conservation among P2Y receptors and the role of some specific residues in sugar recognition seems convincing and the data are conveniently presented.
Finally, the design of ADP-sugars that activate the P2Y12 receptor, based on the transferability of the observations with the UDP-sugars for the P2Y14 receptor, is a first indication that such a recognition is possible and should happen in an analogous binding region. However, the low potencies exhibited by the ATP-sugars, in the micromolar range, are too far from the ATP agonist and the relevance of this mechanism remains to be proved. The difference between P2Y12 and P2T14, with the last one showing much higher potencies for UDP-sugar derivatives than P2Y12 for the corresponding ADP-sugars, remains an interesting question not explored in this manuscript.
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Reviewer #2 (Public Review):
The manuscript employs multiple approaches, including molecular docking, molecular dynamic simulations, and functional experiments to uncover a distinct uridine diphosphate-sugar-binding site on P2Y14 - a key drug target for inflammation and immune responses. Overall, the manuscript is clearly written and the experimental techniques are well-documented. However, it may benefit from further analysis, particularly in terms of validating the binding pose.
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