Improved ANAP incorporation and VCF analysis reveal details of P2X7 current facilitation and a limited conformational interplay between ATP binding and the intracellular ballast domain
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eLife assessment
This manuscript constitutes an important foray into the conformational rearrangements throughout various domains of the notoriously difficult-to-study P2X7 receptor, with a focus on the enigmatic intracellular 'ballast' domain. This is of broad interest to those studying the role of enzymatically active intracellular domains of membrane proteins. The authors provide convincing evidence that the ballast domain is unlikely to undergo major conformational changes upon ATP-induced gating, but additional experimental support is required on the facilitation process and to elucidate the consequences exerted by intracellular factors.
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Abstract
The large intracellular C-terminus of the pro-inflammatory P2X7 ion channel receptor (P2X7R) is associated with diverse P2X7R-specific functions. Cryo-EM structures of the closed and ATP-bound open full-length P2X7R recently identified a membrane-associated anchoring domain, an open-state stabilizing “cap” domain, and a globular “ballast domain” containing GTP/GDP and dinuclear Zn 2+ -binding sites with unknown functions. To investigate protein dynamics during channel activation, we improved incorporation of the environment-sensitive fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)–2-aminopropanoic acid (ANAP) into Xenopus laevis oocyte-expressed P2X7Rs and performed voltage clamp fluorometry. While we confirmed predicted conformational changes within the extracellular and the transmembrane domains, only 3 out of 41 mutants containing ANAP in the C-terminal domain resulted in ATP-induced fluorescence changes. We conclude that the ballast domain functions rather independently from the extracellular ATP binding domain and might require activation by additional ligands and/or protein interactions. Novel tools to study these are presented.
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Author Response
Reviewer #1 (Public Review):
Voltage-clamp fluorometry combines electrophysiology, reporting on channel opening, with a fluorescence signal reporting on local conformational changes. Classically, fluorescence changes are reported by an organic fluoropohore tethered to the receptor thanks to the cysteine chemistry. However, this classical approach does not allow fluorescent labeling of solvent-inaccessible regions or cytoplasmic regions. Incorporation of the fluorescent unnatural amino acid ANAP directly in the sequence of the protein allows counteracting these limitations. However, expression of ANAP-containing receptors is usually weak, leading to very small ANAP-related fluorescence changes (ΔFs).
In this paper, the authors developed an improved method for expression of full-length, ANAP-mutated proteins in …
Author Response
Reviewer #1 (Public Review):
Voltage-clamp fluorometry combines electrophysiology, reporting on channel opening, with a fluorescence signal reporting on local conformational changes. Classically, fluorescence changes are reported by an organic fluoropohore tethered to the receptor thanks to the cysteine chemistry. However, this classical approach does not allow fluorescent labeling of solvent-inaccessible regions or cytoplasmic regions. Incorporation of the fluorescent unnatural amino acid ANAP directly in the sequence of the protein allows counteracting these limitations. However, expression of ANAP-containing receptors is usually weak, leading to very small ANAP-related fluorescence changes (ΔFs).
In this paper, the authors developed an improved method for expression of full-length, ANAP-mutated proteins in Xenopus oocytes. In particular, they managed to increase the ratio of full-length over truncated proteins for C-terminal ANAP incorporation sites. Since C-terminally truncated P2X receptors are usually functional, it is important to maximize the full-length over truncated protein ratio to have a good correspondence between the observed current and fluorescence. Using their improved strategy, they screened for ANAP incorporation sites and ATP-mediated ANAP ΔFs along the whole structure of the P2X7 receptor: extracellular ligand binding domain (head domain), M2 transmembrane segment (gate), as well as a large extracellular domain specific for the P2X7 subtype, the "ballast" domain. The functional role of this domain and its motions following ATP application are indeed unknown. Monitoring ANAP fluorescence changes in this region following ATP binding provides a unique way to study those questions. By analyzing ATP-induced ΔFs from different parts of the receptors, the authors conclude that the ATP-binding domain mainly follows gating, while intracellular "ballast" motions are largely decoupled from ATP-binding
Strengths of the paper:
This paper provides an improved method for efficient unnatural amino acid incorporation in Xenopus oocytes. Thanks to this technique, they managed to enhance membrane expression of ANAP-mutated P2X7 receptors and observed strong fluorescent changes upon ATP application. The paper furthermore describes an impressive screen of ANAP-incorporation sites along the whole protein sequence, which allows them to monitor conformational changes of solvent-inaccessible regions (transmembrane domains) and cytoplasmic regions that were not accessible to cysteine-reactive fluorophores. This screen was performed in a very thorough manner, each ANAP mutant being characterized biochemically for membrane expression, as well as in term of fluorescence changes. The limitations of the approach -small ΔF upon ATP application on wt receptors, problem of baseline fluorescence variations in presence of calcium- are well explained. Overall, this study should thus not only serve as a guide to anyone willing to perform VCF on P2X7 receptors but it should be useful to the whole community of researchers using unnatural amino acids. Thanks to orthogonal labeling with TMRM and ANAP, the authors managed to simultaneously monitor the motions of the extracellular and intracellular domains of P2X7. Finally, they propose methods to simultaneously monitor intracellular domain motion and downstream signaling.
Weaknesses:
Although the fluorescence screen is impressive and well conducted, the biological conclusions remain superficial at this stage. The paper furthermore lacks quantitative analysis. Finally, the title only reflects a minor part of the paper and is therefore not representative of the paper content.
Quantitative analyses (DRCs and current rise times) were now added for the key mutations. In addition, we performed a variety of experiments to address the challenging question of mechanistic insight (mutants that track facilitation) and effects of intracellular factors (mutation of calmodulin binding site, FRET experiments with calmodulin). These data confirmed that deletion of a cysteine-rich intracellular region eliminates current facilitation (Roger et al., 2010) and that some of our mutants indeed track facilitation. However, mutation of the CaM binding site and FRET experiments did not support an effect of calmodulin or were inconclusive. As pointed out above, we think that VCF has limited capacity to identify novel biologically relevant consequences of receptor activation but is more suited to determine the sites and dynamics of already defined interactions.
The title was changed to: "Improved ANAP incorporation and VCF analysis reveals details of P2X7 current facilitation and a limited conformational interplay between ATP binding and the intracellular ballast domain"
Reviewer #2 (Public Review):
The authors aimed to elucidate the structural rearrangements and activation mechanisms of P2X7 upon ATP application by voltage clamp fluorometry (VCF) using fluorescent unnatural amino acid (fUAA) and other fluorophores. They improved the fUAA methodology and detected ATP binding evoked changes in the ATP binding region and other regions. They also observed facilitation of fluorescence (F) changes by repeated application of ATP associated with gating. The F change in the cytoplasmic ballast region was minor, and with their experimental data, they discussed this region is involved in activation by other cytoplasmic factors, such as Ca2+.
The strengths of the study are as follows.
(1) fUAA methodology was improved to enable experiments by one time injection to oocytes (Figs. 1 and Suppl).
(2) They performed intensive mutagenesis study of as many as 61 mutants (Figs. 3, 4, 5).
(3) A careful evaluation of the successful Anap incorporation and formation of full length proteins was performed by western blot analysis (Fig. 2).
(4) By three wave lengths F recording, they obtained better information, i.e. they classified the interpretation of F changes to, quenching, dequenching, increase in polarity and decrease in polarity (Fig. 3E).
(5) They detected F changes upon ATP application in various regions of P2X7, but not many in the ballast region, showing that the ballast region is not well involved in the ATP evoked gating.
(6) They analyzed the kinetics of F and current and their changes upon repeated ATP application to approach the known facilitation mechanisms. The data are very interesting. They concluded that it is intrinsic to the P2X7 molecule and that it is associated not with the ATP binding but with the gating process (Figs. 3F, 4D, 6A).
(7) They performed interesting analysis to clarify the mechanisms of activation by cytoplasmic factors, especially Ca2+ entered via P2X7 (Fig. 6).
The weaknesses of the study are as follows.
(1) As both structures of P2X in the open and closed states are already solved, and the ATP binding evoked structural rearrangements from the ATP binding site to the gate are already known in detail. The structural rearrangements detected in the extracellular region (Fig. 3) and TM region (Fig. 4) upon ATP application are just as expected. The impact and scientific merits of this part are rather limited.
We generally agree that the cryo-EM structures clarified basic principles of receptor function. However, considering the specific features of the P2X7 receptor and its likely regulation/modulation by membrane components and environment and the fact that the actual states of the receptor structures (e.g. facilitated or not?) is not known, we think that VCF analysis of its dynamics in a more native cellular environment is still required to confirm the predicted motions and also has the potential to identify details of "P2X7 fine tuning".
(2) The facilitation mechanism is of high interest. The authors showed it is intrinsic to P2X2 and associated with the gating rather than ATP binding. However, this reviewer cannot have better understanding about the actual mechanism. (a) What is the mechanistic trigger of facilitation? Possibilities are discussed, but it appears there is no clear answer with experimental evidences yet. (b) How is the memory of the 1st ATP application stored in the molecule, i.e. how does the P2X7 structure just before the 1st application differ from that just before the 2nd application of ATP?
These are indeed fundamental questions but based on the available information we do not see a rational approach to address this issue any further. Additional extensive "screening" for ideal fluorophore positions would probably be required and is beyond our possibilities in the present study.
(3) The structural rearrangement of the CaM-M13 region (Fig. 6B, C) attached at the C-terminus by Ca2+ influx through P2X7 upon ATP application is natural due course and not very surprising. Also, it is not accepted as an evidence proving that Ca2+ is the mediator of facilitation.
We apologize, this is a misunderstanding. We only provided protocols for parallel recordings of ANAP with other fluorophores for further analysis of downstream signaling pathways but we did not show or propose any functional consequences of the Ca2+ influx (see also point 7 above).
(4) As to the ballast region, data showed its limited involvement in the ATP-induced structural rearrangements. The function of the ballast region is not clear yet. A possible involvement in GDP binding and/ or metabolism is discussed, but there is no clear experimental evidence.
We are aware of these limitations. In the absence of a clear fluorescence change around the GTP/GDP-binding site or information about its role, it is difficult to investigate its molecular function by VCF. The fact, that (un-)binding of the guanosine nucleotide does not seem to be related to channel opening (McCarthy et al., 2019) further limits our options to study its function and currently it is not even known whether GDP/GTP has just a structural role. However, we identified A564* as a potential reporter for yet undefined processes that might affect GTP/GDP binding and/or metabolism.
Reviewer #3 (Public Review):
This research contributes to optimizing the amber stop-codon suppression protocol for voltage-clamp fluorometry (VCF) experiments using Xenopus oocyte heterologous expression system. By in vitro RNA synthesizing the tRNA and tRNA synthetases, combined with the dominant-negative release factor initially developed by Jason Chin's lab, L-Anap can be site-specifically labeled to proteins by a single microinjection of a mixture of molecular components into the cytoplasm of oocytes. Although it avoids nuclear microinjection to oocytes, it adds more RNA synthesis steps. This strategy of using eRF dominant negative variant (eRF1-E55D), was previously applied to the Anap incorporation system using mammalian cell lines and model proteins (Gordon et al, eLife, 2018). In this previous 2018 paper, with eRF1-E55D, the percentage of full-length protein expression increased substantially. Using oocytes in this paper, this percentage apparently did not increase significantly as shown in Fig. 1D, different from the previous paper. Nevertheless, the overall expression level increased successfully by this method, which could facilitate macroscopic fluorescence measurements, especially considering that L-Anap is relatively dim as a fluorophore.
Anap fluorescence change was measured mostly using its environmental sensitivity, which has limited information in interpreting structural changes. The structural mechanisms proposed could be potentially strengthened and the conclusions could be further validated by combining FRET or other distance ruler experiments with the VCF method. The engineered CaM-M13 FRET experiments mostly report the calcium entry, not measuring the rearrangements of P2X7 directly.
We tried FRET analyses with ANAP-labeled P2X7 and mNeonGreen-labeled CaM but unfortunately, results were inconclusive.
In addition, results of ATP dose-response relationship for channel activation correlated with ATP dose-dependent Anap fluorescence change, especially for sites showing a large percentage of ATP-induced change in fluorescence, would provide more insights regarding the allosteric mechanism of the channel.
We agree, but unfortunately, bleaching of ANAP and the variation of background fluorescence in individual oocytes prevented such analyses .
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eLife assessment
This manuscript constitutes an important foray into the conformational rearrangements throughout various domains of the notoriously difficult-to-study P2X7 receptor, with a focus on the enigmatic intracellular 'ballast' domain. This is of broad interest to those studying the role of enzymatically active intracellular domains of membrane proteins. The authors provide convincing evidence that the ballast domain is unlikely to undergo major conformational changes upon ATP-induced gating, but additional experimental support is required on the facilitation process and to elucidate the consequences exerted by intracellular factors.
-
Reviewer #1 (Public Review):
Voltage-clamp fluorometry combines electrophysiology, reporting on channel opening, with a fluorescence signal reporting on local conformational changes. Classically, fluorescence changes are reported by an organic fluoropohore tethered to the receptor thanks to the cysteine chemistry. However, this classical approach does not allow fluorescent labeling of solvent-inaccessible regions or cytoplasmic regions. Incorporation of the fluorescent unnatural amino acid ANAP directly in the sequence of the protein allows counteracting these limitations. However, expression of ANAP-containing receptors is usually weak, leading to very small ANAP-related fluorescence changes (ΔFs).
In this paper, the authors developed an improved method for expression of full-length, ANAP-mutated proteins in Xenopus oocytes. In …
Reviewer #1 (Public Review):
Voltage-clamp fluorometry combines electrophysiology, reporting on channel opening, with a fluorescence signal reporting on local conformational changes. Classically, fluorescence changes are reported by an organic fluoropohore tethered to the receptor thanks to the cysteine chemistry. However, this classical approach does not allow fluorescent labeling of solvent-inaccessible regions or cytoplasmic regions. Incorporation of the fluorescent unnatural amino acid ANAP directly in the sequence of the protein allows counteracting these limitations. However, expression of ANAP-containing receptors is usually weak, leading to very small ANAP-related fluorescence changes (ΔFs).
In this paper, the authors developed an improved method for expression of full-length, ANAP-mutated proteins in Xenopus oocytes. In particular, they managed to increase the ratio of full-length over truncated proteins for C-terminal ANAP incorporation sites. Since C-terminally truncated P2X receptors are usually functional, it is important to maximize the full-length over truncated protein ratio to have a good correspondence between the observed current and fluorescence. Using their improved strategy, they screened for ANAP incorporation sites and ATP-mediated ANAP ΔFs along the whole structure of the P2X7 receptor: extracellular ligand binding domain (head domain), M2 transmembrane segment (gate), as well as a large extracellular domain specific for the P2X7 subtype, the "ballast" domain. The functional role of this domain and its motions following ATP application are indeed unknown. Monitoring ANAP fluorescence changes in this region following ATP binding provides a unique way to study those questions. By analyzing ATP-induced ΔFs from different parts of the receptors, the authors conclude that the ATP-binding domain mainly follows gating, while intracellular "ballast" motions are largely decoupled from ATP-binding
Strengths of the paper:
This paper provides an improved method for efficient unnatural amino acid incorporation in Xenopus oocytes. Thanks to this technique, they managed to enhance membrane expression of ANAP-mutated P2X7 receptors and observed strong fluorescent changes upon ATP application. The paper furthermore describes an impressive screen of ANAP-incorporation sites along the whole protein sequence, which allows them to monitor conformational changes of solvent-inaccessible regions (transmembrane domains) and cytoplasmic regions that were not accessible to cysteine-reactive fluorophores. This screen was performed in a very thorough manner, each ANAP mutant being characterized biochemically for membrane expression, as well as in term of fluorescence changes. The limitations of the approach -small ΔF upon ATP application on wt receptors, problem of baseline fluorescence variations in presence of calcium- are well explained. Overall, this study should thus not only serve as a guide to anyone willing to perform VCF on P2X7 receptors but it should be useful to the whole community of researchers using unnatural amino acids. Thanks to orthogonal labeling with TMRM and ANAP, the authors managed to simultaneously monitor the motions of the extracellular and intracellular domains of P2X7. Finally, they propose methods to simultaneously monitor intracellular domain motion and downstream signaling.Weaknesses:
Although the fluorescence screen is impressive and well conducted, the biological conclusions remain superficial at this stage. The paper furthermore lacks quantitative analysis. Finally, the title only reflects a minor part of the paper and is therefore not representative of the paper content. -
Reviewer #2 (Public Review):
The authors aimed to elucidate the structural rearrangements and activation mechanisms of P2X7 upon ATP application by voltage clamp fluorometry (VCF) using fluorescent unnatural amino acid (fUAA) and other fluorophores. They improved the fUAA methodology and detected ATP binding evoked changes in the ATP binding region and other regions. They also observed facilitation of fluorescence (F) changes by repeated application of ATP associated with gating. The F change in the cytoplasmic ballast region was minor, and with their experimental data, they discussed this region is involved in activation by other cytoplasmic factors, such as Ca2+.
The strengths of the study are as follows.
(1) fUAA methodology was improved to enable experiments by one time injection to oocytes (Figs. 1 and Suppl).
(2) They performed …Reviewer #2 (Public Review):
The authors aimed to elucidate the structural rearrangements and activation mechanisms of P2X7 upon ATP application by voltage clamp fluorometry (VCF) using fluorescent unnatural amino acid (fUAA) and other fluorophores. They improved the fUAA methodology and detected ATP binding evoked changes in the ATP binding region and other regions. They also observed facilitation of fluorescence (F) changes by repeated application of ATP associated with gating. The F change in the cytoplasmic ballast region was minor, and with their experimental data, they discussed this region is involved in activation by other cytoplasmic factors, such as Ca2+.
The strengths of the study are as follows.
(1) fUAA methodology was improved to enable experiments by one time injection to oocytes (Figs. 1 and Suppl).
(2) They performed intensive mutagenesis study of as many as 61 mutants (Figs. 3, 4, 5).
(3) A careful evaluation of the successful Anap incorporation and formation of full length proteins was performed by western blot analysis (Fig. 2).
(4) By three wave lengths F recording, they obtained better information, i.e. they classified the interpretation of F changes to, quenching, dequenching, increase in polarity and decrease in polarity (Fig. 3E).
(5) They detected F changes upon ATP application in various regions of P2X7, but not many in the ballast region, showing that the ballast region is not well involved in the ATP evoked gating.
(6) They analyzed the kinetics of F and current and their changes upon repeated ATP application to approach the known facilitation mechanisms. The data are very interesting. They concluded that it is intrinsic to the P2X7 molecule and that it is associated not with the ATP binding but with the gating process (Figs. 3F, 4D, 6A).
(7) They performed interesting analysis to clarify the mechanisms of activation by cytoplasmic factors, especially Ca2+ entered via P2X7 (Fig. 6).The weaknesses of the study are as follows.
(1) As both structures of P2X in the open and closed states are already solved, and the ATP binding evoked structural rearrangements from the ATP binding site to the gate are already known in detail. The structural rearrangements detected in the extracellular region (Fig. 3) and TM region (Fig. 4) upon ATP application are just as expected. The impact and scientific merits of this part are rather limited.
(2) The facilitation mechanism is of high interest. The authors showed it is intrinsic to P2X2 and associated with the gating rather than ATP binding. However, this reviewer cannot have better understanding about the actual mechanism. (a) What is the mechanistic trigger of facilitation? Possibilities are discussed, but it appears there is no clear answer with experimental evidences yet. (b) How is the memory of the 1st ATP application stored in the molecule, i.e. how does the P2X7 structure just before the 1st application differ from that just before the 2nd application of ATP?
(3) The structural rearrangement of the CaM-M13 region (Fig. 6B, C) attached at the C-terminus by Ca2+ influx through P2X7 upon ATP application is natural due course and not very surprising. Also, it is not accepted as an evidence proving that Ca2+ is the mediator of facilitation.
(4) As to the ballast region, data showed its limited involvement in the ATP-induced structural rearrangements. The function of the ballast region is not clear yet. A possible involvement in GDP binding and/ or metabolism is discussed, but there is no clear experimental evidence. -
Reviewer #3 (Public Review):
This research contributes to optimizing the amber stop-codon suppression protocol for voltage-clamp fluorometry (VCF) experiments using Xenopus oocyte heterologous expression system. By in vitro RNA synthesizing the tRNA and tRNA synthetases, combined with the dominant-negative release factor initially developed by Jason Chin's lab, L-Anap can be site-specifically labeled to proteins by a single microinjection of a mixture of molecular components into the cytoplasm of oocytes. Although it avoids nuclear microinjection to oocytes, it adds more RNA synthesis steps. This strategy of using eRF dominant negative variant (eRF1-E55D), was previously applied to the Anap incorporation system using mammalian cell lines and model proteins (Gordon et al, eLife, 2018). In this previous 2018 paper, with eRF1-E55D, the …
Reviewer #3 (Public Review):
This research contributes to optimizing the amber stop-codon suppression protocol for voltage-clamp fluorometry (VCF) experiments using Xenopus oocyte heterologous expression system. By in vitro RNA synthesizing the tRNA and tRNA synthetases, combined with the dominant-negative release factor initially developed by Jason Chin's lab, L-Anap can be site-specifically labeled to proteins by a single microinjection of a mixture of molecular components into the cytoplasm of oocytes. Although it avoids nuclear microinjection to oocytes, it adds more RNA synthesis steps. This strategy of using eRF dominant negative variant (eRF1-E55D), was previously applied to the Anap incorporation system using mammalian cell lines and model proteins (Gordon et al, eLife, 2018). In this previous 2018 paper, with eRF1-E55D, the percentage of full-length protein expression increased substantially. Using oocytes in this paper, this percentage apparently did not increase significantly as shown in Fig. 1D, different from the previous paper. Nevertheless, the overall expression level increased successfully by this method, which could facilitate macroscopic fluorescence measurements, especially considering that L-Anap is relatively dim as a fluorophore.
Anap fluorescence change was measured mostly using its environmental sensitivity, which has limited information in interpreting structural changes. The structural mechanisms proposed could be potentially strengthened and the conclusions could be further validated by combining FRET or other distance ruler experiments with the VCF method. The engineered CaM-M13 FRET experiments mostly report the calcium entry, not measuring the rearrangements of P2X7 directly. In addition, results of ATP dose-response relationship for channel activation correlated with ATP dose-dependent Anap fluorescence change, especially for sites showing a large percentage of ATP-induced change in fluorescence, would provide more insights regarding the allosteric mechanism of the channel.
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