To investigate protein dynamics during channel activation, we improved incorporation of the environment-sensitive fluorescent unnatural amino acid L-3-–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.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 A plasmid encoding an ANAP-specific bio-orthogonal suppressor tRNA/aminoacyl-tRNA synthetase pair (P2X receptors (P2XR) are trimeric non-selective cation channels that are activated by extracellular adenosine triphosphate (ATP). The structure of a single P2X subunit has been compared to a dolphin, with two transmembrane domains (TM1 and TM2) that form the ‘fluke’, and a large extracellular domain, comprising the ‘body’, left and right ‘flippers’, and a ‘head’ domain that reaches over one of three inter-subunit ATP binding sites . The intase pair  has beenase pair  and was ase pair . This stase pair .Xenopus laevis oocyte-expressed proteins and analyzed membrane surface expression and functionality for a total of 61 P2X7R mutants with ANAP substitutions in the extracellular head domain, the second transmembrane domain (TM2), and the intracellular N- and C-termini. Using VCF, we identified 19 positions in which ANAP reported ATP-induced localized rearrangements. To further expand the VCF toolbox, we demonstrate simultaneous recordings of fluorescence changes from ANAP in combination with other fluorophores. We conclude from our data that (i) current facilitation is intrinsic to the P2X7 protein and likely caused by a change in gating and (ii) the cytoplasmic ballast functions rather independently from the extracellular ligand binding domain and might require activation by additional ligands or protein interactions.Here, we provide an improved method for fUAA incorporation into Xenopus oocyte-expressed protein, we initially used the P2X1R as it was already intensively studied in our lab  protein  and ballast (I577*) domains, while it was most efficient for ANAP-substitutions in positions 517–537 (in particular L527* and E537*) and in the very C-terminus (Y595* and 596*).Starting from T357 in the C-terminus, introduction of the In summary, most substitutions within the C-terminus led to a dominant formation of truncated P2X7 protein besides full-length receptors. Nevertheless, the majority of these constructs showed clear current responses , Table 1Next, we recorded ANAP fluorescence changes upon application of 0.3 mM ATP. Control oocytes expressing wt P2X7R showed a gradual fluorescence decrease during ATP application, even when no ANAP was injected . A similAs a prodan derivative, ANAP is highly sensitive to the polarity of its environment and shows a redshift in emission with increasing polarity . ConsequTo allow differentiation between presumably wavelength-independent (de-)quenching of ANAP fluorescence by other molecules or spectral shifts due to changes in the polarity, we simultaneously recorded fluorescence in distinct spectral segments, i.e., (i) 430–470 nm and 470–500 nm with filter set 1 and (ii) 430–490 nm and >500 nm with filter set 2 . This alWe first recorded ATP-induced fluorescence changes from P2X7Rs containing ANAP in the head domain, which projects over the ATP-binding site , since this revealed more pronounced signals for most mutants.To exclude fluorescence changes induced by a direct interaction with ATP and to further investigate P2X7 gating, we next investigated positions 339–341 , just prNotably, fluorescence signals from P2X7 T340* were also significantly larger during the first ATP application compared to the second , suggest15, which is part of the cytoplasmic cap structure , and upstream of the cytosolic ballast domain (C388* and V392*). Surface expression of functional full-length receptors was observed for all constructs except for K387* and C388*. In contrast to the juxtamembrane N-terminal residues, however, only one of these C-terminal mutants, T361*, showed a fluorescence change, albeit in only ~50% of the recordings  did not form functional receptors at all. For most of the remaining constructs, no specific fluorescence changes could be detected, despite promising surface transport and current responses comparable to wt receptors for at least 12 of them see .50 values similar to wt P2X7 and were not functional in control oocytes injected without ANAP  binding motif   . To iden41-S560)  as well 41-S560)  on ANAP 41-S560) . This arTaken together, only two positions, D423 and A564, could be identified within the ballast domain, where ANAP reported environmental changes, suggesting only limited ATP-induced movements in this domain. However, mutant A564* has great potential as a reporter for yet undefined processes that affect GDP binding and/or metabolism.Based on the above findings, we propose that yet unknown intracellular ligands or protein interactors are required to mediate downstream signaling via the ballast domain. As potential tools to further investigate such molecules and the dynamics of their molecular interplay with the P2X7R, we combined ANAP with other fluorophores and equipped the VCF setup with a second LED for parallel excitation of two different fluorophores within the same protein.50 values for ATP at these mutants and at wt P2X7 were comparable  suited to investigate the dynamics of P2X7 activation in different parts of the receptor by parallel labeling with the thiol-reactive fluorophore TMRM in the extracellular head domain and with ANAP in the cytoplasmic N-terminus. As seen in mparable .2+, an important mediator of intracellular signaling, we also established a protocol to combine VCF recording of ANAP-fluorescence with imaging of P2X7-mediated Ca2+ influx by fusing the genetically encoded Ca2+-sensor R-GECO1.2    has alsointeins) . While cinteins) , expressinteins) . Here, wexpanded . Here, w- channel antagonists  , palmitoagonists , and it litation . Here, wannexins . InteresFluorescence signals recorded from ANAP in positions near the channel gate  could reBased on the above observations, we propose that the faster activation upon the second ATP application is an intrinsic property of the P2X7R. This conclusion is also in good agreement with the fact that the current facilitation but not downstream signaling events is seen in truncated P2X7 constructs . One posWhile the functionality of P2X7 as a cation channel is not impaired by lack of the intracellular C-terminus , its delXenopus oocyte-expressed protein and performed an extensive VCF analysis of P2X7R mutants carrying ANAP in 61 positions throughout the receptor. We conclude from our data, that current facilitation is, at least partly, an intrinsic property of the P2X7R and involves an accelerated channel gating rather than ligand binding. In addition, we propose that ligand-induced extracellular and TM domain movements are not significantly translated to the cytosolic ballast domain and that intracellular ligands or interactors are required to ‘activate’ this domain. Protocols for simultaneous recording of ANAP with TMRM, Ca2+-dependent R-GECO1.2, or mNeonGreen-labeled FRET partners are presented that might help to validate P2X7 downstream signaling events and analyze their molecular mechanisms and dynamics, once such interactors have been reliably determined.In summary, we improved ANAP incorporation into X. laevis females were obtained from NASCO  and kept at the Core Facility Animal Models (CAM) of the Biomedical Center (BMC) of LMU Munich, Germany (Az:4.3.2–5682/LMU/BMC/CAM) in accordance with the EU Animal Welfare Act. To obtain oocytes, frogs were deeply anesthetized in MS222 and killed by decapitation. Surgically extracted ovary lobes were divided into smaller lobes and dissociated by ~2.5 hr incubation (16°C) with gentle shaking in ND96 solution  containing 2 mg/ml collagenase  and subsequently defolliculated by washing (15 min) with Ca2+-free oocyte Ringer solution . Stage V-VI oocytes were selected and kept in ND96 containing 5 µg/ml gentamicin until further use. In some cases, oocytes were commercially obtained , or ovaries were provided by Prof. Dr. Luis Pardo .N-terminally His-tagged rat P2X1 cDNA in pNKS2 has been described . An EGFPXenopus globin 5’-UTR, and a Kozak sequence . pUC19o was generated by insertion (from 5` to 3`) of a synthesized T7 promoter sequence, a sequence  (GeneArtsequence  followedsequence  (for detThe cDNA sequence of the aminoacyl-tRNA synthetase was obtained from the plasmid pANAP (Addgene #48696)  and subcX. laevis eRF1 (NCBI Reference Sequence: NM_001090894.1) with an E55D mutation  was cloned into pNKS2 via Gibson assembly. For recombinant expression in E. coli, the coding sequence of His-eRF1(E55D) was cloned into a modified pET28a vector via Gibson assembly.The coding sequence of Site-specific mutagenesis was performed with the Q5 Site-Directed Mutagenesis Kit (based on PCR-amplification) according to the manufacturer’s protocol . Oligonucleotides were ordered from metabion GmbH .ochre (TAA) or opal (TGA) stop codon for normal translational termination to avoid C-terminal ANAP incorporation and read-through and were confirmed by sequencing .All constructs contained either an 2, 10% (v/v) glycerol, 0.1% (v/v) Triton X-100, 10 µg/ml DNase I, 100 µg/ml lysozyme), and sonicated  for 5 min in an ice bath. The lysate was pelleted at 40,000 × g (1 hr at 4°C). The supernatant was filtered (0.2 µm) and applied onto a Ni-NTA column . Bound protein was washed with 10 column volumes of washing buffer  and eluted with 6 column volumes of elution buffer . The eluate was concentrated , and buffer was exchanged by low-salt buffer  for subsequent anion exchange chromatography on a 5 ml Mono-Q column . Following an elution gradient with high-salt buffer , protein-containing fractions were pooled, concentrated, and buffer was exchanged (1× PBS with 500 mM NaCl) for size exclusion chromatography on a Superdex 75 Increase (10/300). Purified His-eRF1(E55D) was shock-frozen in 10 µl aliquots and stored at –80°C.NiCo(DE3) bacteria were transformed with His-eRF1(E55D) in pET28a. 5 ml of a LB-Kanamicin pre-culture (~12 hr) was added to 300 ml ZY-5052 autoinduction media  supplemeTo prepare templates for cRNA synthesis, plasmids were linearized with EcoRI-HF (pNKS2) or NotI-HF (pUC19o) from New England Biolabs GmbH  and purified via MinElute Reaction Cleanup columns  according to the manufacturer’s protocol. Alternatively, templates  and the 3’-terminal poly A) were amplified by PCR and purified using the NucleoSpin Gel and PCR Clean-up Kit  according to the manufacturer’s protocol.Capped cRNA was synthesized using the mMESSAGE mMACHINE SP6 or T7 Transcription Kits , precipitated with LiCl, and dissolved in nuclease-free water (1 µg/µl if not stated otherwise).amber suppressor tRNA sequence was translated from the plasmid pANAP (Addgene #48696) (The  #48696) , provideA Nanoject II injector  was used for nuclear and cytoplasmic injections.cRNAs encoding cysteine-substituted receptors for TMRM labeling were injected as described . Two difamber suppressor tRNA/tRNA synthetase pair . 50.6 nl per oocyte were injected into the cytoplasm. Uninjected oocytes and oocytes injected with wt receptor cRNA served as negative and positive controls, respectively. Nuclease free water served as a substitute for individual components in control groups.The 1-step injection method was performed as described before  with addX. laevis oocyte-expressed receptors, different procedures, concentrations of substances, and injection time points were compared  supplemented with gentamicin (50 µg/ml) at 16°C for at least 2 days.Injected oocytes were kept in ND96 . Membrane proteins were extracted by 10 min incubation on ice and separated from the debris by two centrifugation steps . 100 µl of the protein extract were then supplemented with 400 µl of homogenization buffer containing 10 mM imidazole and added to 50 µl Ni2+-NTA agarose beads  preconditioned with washing buffer . After 1 hr incubation under inversion at 4°C in the dark, beads were washed three to four times with 500 µl washing buffer, and His-tagged protein was eluted  with 2×50 µl elution buffer . 32 µl of the eluate were supplemented with 8 µl 5× lithium dodecyl sulfate (LiDS) sample buffer , incubated at 95°C for 10 min, and separated by reducing SDS-PAGE on an 8% gel. Fluorescence-labeled protein was visualized with a Typhoon trio fluorescence scanner , and relative protein quantities were determined using FIJI  in 200 μl 0.003% (m/V) aminoreactive, membrane-impermeant Cy5 Mono NHS Ester  in ND96  and then washed in ND96. Bright blue-stained damaged oocytes were then discarded, and intact oocytes were homogenized with a 200 μl pipet tip in 10 µl homogenization buffer per oocyte  complemented with flufenamic acid and ethylene glycol tetraacetic acid (EGTA) (both 0.1 mM). For measurements with Ca2+-containing buffers, EGTA was omitted, and Ca2+ (0.2–0.5 mM) was added . If not otherwise noted, the agonist solution contained 300 μM ATP and was applied for 15 s in 195 s intervals. Intracellular electrode resistances were below 1.2 MΩ, and recordings were performed at room temperature at a holding potential of –30 mV to keep the current amplitudes reproducible. The solution exchange in the lower chamber is finished in about 1 s . Since UV excitation in oocytes causes relatively high background fluorescence levels, detectors must feature a wide dynamic range, while maintaining a sufficiently high sensitivity in order to record small fluorescence changes. To this end, two cooled, high-sensitivity MPPC detectors  were used for simultaneous fluorescence detection at two different spectral segments. For optical filters and dichroic mirrors see Key resource table.Single-channel fully programmable instrumentation amplifiers with Bessel low-pass filter characteristics  were used for signal scaling. To minimize photobleaching, LEDs were pulsed using self-developed high-speed LED drivers with sub-μs rise time. Pulse lengths were set in the ~20 μs range to allow for the fluorescence readout signal chain to settle. Fluorescence signal digitization was synchronized to the excitation pulses using an STM32F407 microcontroller . Its timer peripherals were re-triggered by each ADC conversion cycle in order to create an LED illumination pulse that starts shortly before the next ADC conversion cycle. Whenever two excitation wavelengths were used, excitation pulses were staggered in time with the longer wavelength excitation pulse signal being digitized first, preventing bleedthrough of background fluorescence excited by the shorter excitation wavelength to the longer-wavelength detection channel. A water-immersion objective with high numerical aperture and a large working distance  was used to maximize the collection of emitted photons and to focus on the oocyte membrane.Currents were measured with a Turbo Tec-05X amplifier and CellWorks E 5.5.1 software  and were used for current and fluorescence recordings and valve control. Current signals were digitized at 400 Hz and downsampled in CellWorks to 200 Hz.Ref) of 300 μM was applied until stable responses were obtained and was then alternately applied with ATP concentrations ranging from 10 μM to 3 mM (ATPTest). All responses were normalized to the response of ATPRef, and EC50 values were calculated using the four-parameter Hill equation: 50−X)*nH% Response = Bottom + (Top−Bottom)/(1+10^[(LogEC]) with Bottom and Top constrained to 0%, and maximum responses, respectively, X corresponding to the log of agonist concentration, and Hn corresponding to the Hill coefficient.To determine agonist dose-response curves, ATP was applied for 15 s in 195 s intervals. A reference concentration : Fluorescence signals were denoised using a fifth-order Bessel filter with a low-pass corner frequency of 4 Hz. Maximum amplitudes of ATP-evoked current and fluorescence responses from different receptor constructs were summarized, compared, and visualized using GraphPad Prism software . The following inclusion criteria were applied for recordings:(i) ATP application must evoke a current response >0.1 μA, (ii) leak currents must be stable for the duration of the recording (at least two ATP applications), (iii) repeated ATP applications must elicit reproducible current responses (>0.8 μA), (iv) fluorescence signals must be without mechanical artifacts and clearly distinguishable from fluorescence changes of wt expressing oocytes (see below). 2–3 days after injection, repeated application of 300 μM ATP to wt-expressing oocytes elicited reproducible currents , which were taken as a reference. Longer expression times resulted in irregular and irreproducible current responses and less stable oocytes. In case of mutated receptors, longer expression times were often needed to yield current responses comparable to wt P2X7.We observed a gradual decrease in fluorescence signal for the duration of ligand application in control oocytes expressing wt receptors even in the absence of ANAP. To distinguish ANAP-specific fluorescence signals from these gradual changes, for signal analysis only fluorescence changes upon ATP application were considered that were either positive, or negative but additionally not linear. If fluorescence signals from mutant expressing oocytes were not distinguishable from fluorescence changes observed for wt expressing oocytes no fluorescence change was assumed (0% ΔF/F). Only signals that were recorded in at least three different oocytes were considered for analysis. Additionally, fluorescence changes that were recorded in less than 40% of analyzed oocytes expressing one specific receptor construct or that had averaged ΔF/F values <0.3% were not considered.t-test or two-tailed paired Student’s t-test, as indicated. Values of p<0.05 were defined as statistically significant with *, **, ***, and **** denoting values of p<0.05, 0.005, 0.0005, and 0.0001 or 0.00005, respectively.Data were either represented as mean ± S.D., as box plots, or as mean ± S.E.M. with the number of recordings given in brackets, and statistical analysis was performed by either two-tailed unpaired Welch’s https://doi.org/10.5061/dryad.p8cz8w9tb). The source data files of All data generated or analyzed during this study are included in the manuscript and supporting files. Original VCF recordings, extracted VCF data, and scans from SDS-PAGE gels are provided as source data files for Note that original current and fluorescence recordings provided as comma separated value files each contain three columns of values (from left to right): (1) current values, (2) fluorescence signals of longer emission wavelengths, and (3) fluorescence signals of shorter emission wavelengths. This manuscript constitutes a valuable 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. The molecular origin of the facilitation process and effects by intracellular factors will require future study, but the authors provide convincing evidence that the ballast domain is unlikely to undergo major conformational changes upon ATP-induced gating. The work is of interest to those interested in the role of enzymatically active intracellular domains of membrane proteins. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Aldrich as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Gucan Dai (Reviewer #3).Thank you for submitting your article "Voltage clamp fluorometry analysis of the P2X7 receptor suggests a limited conformational interplay between extracellular ATP binding and the intracellular ballast domain" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:The reviewers appreciated the effort needed to generate this impressive data set. Although there was enthusiasm for the overall approach and the reviewers agreed on the potential of the study, it was felt that additional mechanistic insight on facilitation and impact of intracellular factors would be required before the work would be of sufficient interest. Therefore we ask you to carefully address the following essential revisions:2+/CaM). This is crucial to provide mechanistic insight on the functional role of the ballast domain.(1) Examination of fluorescence changes in the ballast domain (e.g. A564*) in response to manipulation of at least one or two relevant cytoplasmic factors  or Ca2+ or similar). This is particularly interesting for the intracellular N-terminus, which cannot be labeled with cysteine-reactive fluorophores.(2) Careful validation that the fluorescence changes from e.g. S124*, F11*, S127C do in fact track facilitation. This could be achieved by studying fluorescence signals of mutants that alter or eliminate facilitation (if those mutants exist) or by examining the effect of experimental manipulations of cytoplasmic factors on fluorescence (and current) facilitation  A more thorough characterization of key mutants in terms of ATP sensitivity of current and fluorescence, facilitation and activation/deactivation kinetics. This is required for a more quantitive analysis and has the potential to yield additional insight on the mechanism of receptor activation.Reviewer #1 (Recommendations for the authors):I would have expected the authors to functionally characterize the mutants on which they focus in the paper, for instance by measuring the ATP EC50, to have an idea of whether ANAP incorporation at the different sites perturbs receptor function.When studying fluorescence changes at the level of the head domain (p7), the authors write that "fluorescence and current changes started simultaneously and showed shorter rising times upon repeated ATP applications"; "K127* showed a fluorescence change that was faster than the current increase" but no quantification of the fluorescence and current kinetics is given. It would be nice to have such quantification. It would be also nice to have a comparison of the fluorescence and current ATP dose-response curves for mutants showing sufficiently large fluorescence changes. This would further support the fact that ANAP at the level of the head domain tracks gating.ANAP incorporation at TM2 positions seems to induce constitutive currents in P2X7Rs. Is there a channel blocker specific for P2X7Rs that would allow to give an estimation of constitutive activity of these mutants?Page 9, it is written that "the kinetics of F11* fluorescence correlated with current facilitation". As before, this statement solely relies on the qualitative observation of current and fluorescent traces. Quantitative measurements of fluorescence and current kinetics during the first and second application of ATP is necessary to substantiate that claim. Here again, a dose-response curve of current and fluorescence for ATP of P2X7-F11* would be a plus to determine the gating step(s) tracked by ANAP at this position.Xenopus oocytes, investigate potential ATP-induced currents carried by these truncated receptors and measure ATP dose-response curves. Comparing ATP dose-response curves of ANAP-mutants to purely truncated proteins should allow estimating the contribution of truncated proteins to the general current.Attributing the lack of ATP-induced fluorescence changes observed in mutants with ANAP incorporated in the ballast domain to a lack of functional coupling between the extracellular and the intracellular domains is a strong assumption. The issue with ANAP incorporation in the C-terminus is the membrane expression of truncated proteins that are functional. Since the authors showed that these truncated proteins exists, if they are functional, they contribute to the measured current. The proportion of ANAP-labeled proteins could thus be too low to yield fluorescence changes despite large currents. To study the contribution of truncated proteins, the authors should characterize these truncated proteins by expressing just the mRNA with the amber stop codon in It seems that calcium induces fluctuations of ANAP fluorescence. Does calcium have any direct effect on ANAP fluorescence?It seems that fluorescence changes at some positions of the extracellular head domain and intracellular N-terminus track the process of facilitation. Tracking of this process is probably the most interesting finding of this paper, since the mechanism of facilitation is not known. In the paper, facilitation is measured as the increase of channel opening kinetics upon multiple applications of ATP. However, the first application of ATP clearly shows a small, fast component and a bigger, slow component . Could it be that the first component reflects initial gating and the second one the slow facilitation process that occurs upon prolonged ATP application? Could the fast kinetics of K217* fluorescence match the fast component of current, indicating that K217* tracks the initial ATP opening step but not current facilitation? Similarly, on Figure 6 it looks like TMRM kinetics match first the fast current component, while F11* fluorescence matches the kinetics of current facilitation. Could the authors comment on it?Reviewer #2 (Recommendations for the authors):To strengthen the points of weakness above and to elevate the scientific merits of the paper, I have some comments which require attention.(1A) I am curious to know the P2X7 structural differences by VCF between just before the 1st application and just before the 2nd application of ATP, to clarify the structural background of "memory" of ATP application.2+, i.e. Ca2+ influx via P2X7.(1B) Will there be any difference in (1A), in the presence and absence of extracellular Ca2+/CaM (Lines 335-342), However, there are no experimental results to support or deny them. It is desired to examine the effect of experimental manipulations of these factors on the F(and I) facilitation .(2A) Facilitation of F change was detected e.g. in S124*, F11*, S127C. Possible mechanisms of facilitation are discussed including mediators such as GDP, cholesterol, palmitoylation, Ca(2B) Line 273: "the so-called current facilitation in P2X7 is due to a change in receptor gating rather than ligand binding", Line 366: "Ligand binding is unaltered between the 1st and 2nd activation and consequently, changes in channel gating account for the observed current facilitation". I wonder if this is correct, as F facilitation is observed in S124* which is close to the ATP binding site .(2C) F facilitation was detected not in S124C-TMRM , not in K127* , but in S124* . I cannot fully understand the ground of differences.2+/CaM are discussed (lines 407-415). The authors showed limited structural rearrangements of the ballast domain upon ATP application, but there is no evidence by VCF to prove activation by intracellular ligand. I would like to see data of F change of such as A564* in the ballast domain in response to manipulation of intracellular factors.(3) As to the function of the ballast domain, it is described "intracellular ligand or interactors are required to "activate" this domain" (line 421). A possible involvement of the ballast domain to the functional modulation of P2X7 by the binding of various factors such as GTP/GDP, LPS and Ca(4) Figure 4C: Does the F facilitation in T340* truly reflect "facilitation", in spite of the absence of current facilitation (as described in line 376)? The authors explained that a lack of faster current in the 2nd application is due to "strongly facilitated gating" (line 377). If that is the case, why there is F facilitation?2+?(5) Do the F change of R-Geco  and FRET change by mNeonGreen  disappear in the absence of extracellular CaReviewer #3 (Recommendations for the authors):eLife.37248.1. Please cite this paper when talking about eRF-E55D as well, https://doi.org/10.7554/2. The 3'-terminal CCA-sequence is already in the pANAP plasmid. More elaboration on how it augmented the translation is needed to make it clear to readers.3. Additional FRET experiments might be out of the scope of this paper, but please add more discussion and references regarding this possibility in the paper.4. One area that could be improved on is the uncoupled "ballast" domain. How might GTP/GDP and dinuclear Zn2+-binding rearrange this domain? Can pharmacological manipulations be used to change intracellular GTP/GDP or Zn2+ levels to see how Anap labeled in this domain changes its fluorescence differently? Could the palmitoylation of the anchor domain play a role for uncoupling the "ballast' domain and for the current facilitation?5. Line 634, should be "artifacts"? Essential revisions:The reviewers appreciated the effort needed to generate this impressive data set. Although there was enthusiasm for the overall approach and the reviewers agreed on the potential of the study, it was felt that additional mechanistic insight on facilitation and impact of intracellular factors would be required before the work would be of sufficient interest. Therefore we ask you to carefully address the following essential revisions:2+/CaM). This is crucial to provide mechanistic insight on the functional role of the ballast domain.(1) Examination of fluorescence changes in the ballast domain (e.g. A564*) in response to manipulation of at least one or two relevant cytoplasmic factors  or Ca2+ and GTP has not been defined so far and no functional readouts for mutations in the GTP or Zn2+ binding sites are known, which hampers their investigation. At least in the Zn2+ binding site, we expected mutations to be not tolerated , likely because of a structural function in receptor folding. Also, our set-up and experimental conditions do not allow a direct comparison of effects before and after intracellular modulation (e.g. by Zn2+ or GDP/GTP).We agree that more insight in the role of the ballast domain would be highly informative. However, the relevance of the cytoplasmic factors such as ZnWe therefore concentrated on possible effects of (de-)palmitoylation and a previously described interaction with calmodulin and generated and analyzed the following three additional mutants:(i) Cys-Ala P2X7  in the cysteine-rich region by alanine residues,(ii) DCys P2X7 (deletion of the cysteine-rich intracellular region (S360-C377)), and(iii) DCaM P2X7  binding site by three point mutations I541T, S552C, V559G Roger et al., 2010).According to  and , DCys- and Cys-Ala mutants were expected to be not palmitoylated and to show no facilitation. In agreement with this hypothesis, we indeed did not see facilitation in the DCys mutant while expression of the Cys-Ala mutant was too low for analysis . Expression of these mutants, was further reduced by the additional mutations for ANAP incorporation , making a reliable detection of the corresponding fluorescence changes impossible.The CaM-binding site in rat P2X7 was previously shown to affect current facilitation . Here, the DCaM mutant showed facilitation comparable to wt P2X7. Likewise, combination with ANAP in intracellular positions or the head domain  revealed similar fluorescence changes as observed for the single mutants with intact CaM binding sites. This argues against a major functional effect of the CaM binding site mutation on current kinetics or molecular movements in the oocyte-expressed receptor. These data are now included in Figure 5—figure supplement 3 and mentioned in line 286ff.As an additional approach to address a potential interaction of the P2X7 receptor with CaM, we performed FRET experiments with mNeonGreen-tagged CaM and ANAP-labeled P2X7 . However, these recordings revealed no differences to the negative control (wt P2X7 with co-injected ANAP and mNeonGreen-tagged CaM) and were therefore discontinued.These additional data are now added as Figure 6—figure supplement 3 and referred to in the text (lines 343ff).Although these experiments cannot exclude an interaction with CaM, they do not support an important role of CaM in the observed current facilitation.2+ or similar). This is particularly interesting for the intracellular N-terminus, which cannot be labeled with cysteine-reactive fluorophores.(2) Careful validation that the fluorescence changes from e.g. S124*, F11*, S127C do in fact track facilitation. This could be achieved by studying fluorescence signals of mutants that alter or eliminate facilitation (if those mutants exist) or by examining the effect of experimental manipulations of cytoplasmic factors on fluorescence (and current) facilitation . In agreement, current rise times  of the first and second ATP applications to the DCys mutant were unaltered in our experiments. In contrast, current rise times clearly accelerated in the wt, S124*, and F11* mutations, demonstrating that we can indeed identify current facilitation in our set-up. However, the DCys and Cys-Ala mutants showed already decreased currents and their combination with sites for ANAP incorporation  resulted in further reduced functional expression, which prevented analysis of the corresponding fluorescence changes.An S127C mutant was not prepared or mentioned in the text and we assume that the reviewer referred to K127*, which showed facilitation only for the current but not for the fluorescence. For DCys/K127*, robust fluorescence changes were only detected in one out of 12 oocytes. In this case, the fluorescence changes were virtually identical upon first and second ATP application, as expected.As an alternative approach, we also combined the three ANAP-substituted mutants  with an S23N mutant that has been shown to alter the kinetics of receptor activation in human P2X7 . However, this mutation did not induce an effect on rat P2X7 kinetics.The analyses confirming facilitation of current rise times in the S124*, F11* mutations and elimination of current facilitation by the DCys mutation are now included in Figure 3—figure supplement 3 and in lines 190ff in the text.2+ was generally omitted in the presented VCF recordings since it interfered with ANAP fluorescence measurements . Ca2+-containing buffer affected overall baseline fluorescence in VCF-experiments independent of the presence or absence of ANAP and additionally promoted receptor desensitization , making these conditions unsuitable for reliable detection and analysis of receptor responses.Extracellular Ca2+ on ANAP fluorescence , albeit only at very high concentrations.We now added photometric data, demonstrating also a direct effect of Ca(3) A more thorough characterization of key mutants in terms of ATP sensitivity of current and fluorescence, facilitation and activation/deactivation kinetics. This is required for a more quantitive analysis and has the potential to yield additional insight on the mechanism of receptor activation.We generated ATP dose response curves for the current responses of wt P2X7 receptor, the key mutants F11*, S124*, K127*, D423*, A564*, and the TMRM-labeled F11*/S124C double mutant and show that their ATP sensitivities differed less than 3fold from that of the wt P2X7.Unfortunately, dose response analysis of fluorescence responses could not be performed due to ANAP bleaching during the long recording protocol (about 15-40 min) and the high variability of background fluorescence, which made normalization of responses from different oocytes impossible.For a quantitative analysis of current facilitation/activation kinetics, we determined the 10-50% rise times of current and fluorescent signal increases upon first and second ATP application for wt P2X7, the DCys mutant, the DCaM mutant, as well as mutants F11*, S124*, K127*, T340*, and the TMRM-labeled F11*/S124C double mutant. Kinetics of channel deactivation were not analyzed as they were strongly delayed, most likely due to turbulences in the measuring chamber that resulted in a slow wash-out of ATP.These data are now provided in Figure 3—figure supplement 3, Figure 4—figure supplement 1, Figure 5—figure supplement 2 and 3, and Table 2. For corresponding text see specific reviewer questions below.Reviewer #1 (Recommendations for the authors):I would have expected the authors to functionally characterize the mutants on which they focus in the paper, for instance by measuring the ATP EC50, to have an idea of whether ANAP incorporation at the different sites perturbs receptor function.50 values of current responses have now been added for the wt P2X7 receptor and key mutants F11*, S124*, K127*, D423*, A564* as well as the F11*/S124C double mutant. EC50 values at the mutants differed less than 3-fold from that at the wt. These data have been included in Table 2, Figure 3—figure supplement 3, and Figure 5—figure supplement 2 and were referred to in lines 208, 271, 315.ATP ECWhen studying fluorescence changes at the level of the head domain (p7), the authors write that "fluorescence and current changes started simultaneously and showed shorter rising times upon repeated ATP applications"; "K127* showed a fluorescence change that was faster than the current increase" but no quantification of the fluorescence and current kinetics is given. It would be nice to have such quantification. It would be also nice to have a comparison of the fluorescence and current ATP dose-response curves for mutants showing sufficiently large fluorescence changes. This would further support the fact that ANAP at the level of the head domain tracks gating.ATP dose response curves for currents have been added (see above). Generation of dose response curves for fluorescence was hampered by bleaching and varying background fluorescence of individual oocytes .Quantification of current and fluorescence kinetics for the key mutants F11*, S124*, K127* and the TMRM-labeled F11*/S124C double mutant has now been performed and the respective data have been included in Figure 3—figure supplement 3 .ANAP incorporation at TM2 positions seems to induce constitutive currents in P2X7Rs. Is there a channel blocker specific for P2X7Rs that would allow to give an estimation of constitutive activity of these mutants?To our knowledge, there is no channel blocker of P2X7 available. However, the P2X7 antagonist A438079  was able to decrease the leak currents of the T340* and L341* mutants. These data are now included as Figure 4—figure supplement 1 (referred to in line 223).Page 9, it is written that "the kinetics of F11* fluorescence correlated with current facilitation". As before, this statement solely relies on the qualitative observation of current and fluorescent traces. Quantitative measurements of fluorescence and current kinetics during the first and second application of ATP is necessary to substantiate that claim. Here again, a dose-response curve of current and fluorescence for ATP of P2X7-F11* would be a plus to determine the gating step(s) tracked by ANAP at this position.These data have now been included .Xenopus oocytes, investigate potential ATP-induced currents carried by these truncated receptors and measure ATP dose-response curves. Comparing ATP dose-response curves of ANAP-mutants to purely truncated proteins should allow estimating the contribution of truncated proteins to the general current.Attributing the lack of ATP-induced fluorescence changes observed in mutants with ANAP incorporated in the ballast domain to a lack of functional coupling between the extracellular and the intracellular domains is a strong assumption. The issue with ANAP incorporation in the C-terminus is the membrane expression of truncated proteins that are functional. Since the authors showed that these truncated proteins exists, if they are functional, they contribute to the measured current. The proportion of ANAP-labeled proteins could thus be too low to yield fluorescence changes despite large currents. To study the contribution of truncated proteins, the authors should characterize these truncated proteins by expressing just the mRNA with the amber stop codon in Thank you for the helpful suggestion, these control experiments have now been performed for the D423* or A564* mutants and revealed less than 5% of the respective control current responses  if the respective RNAs were co-injected only with water.50 values at the ANAP-labeled constructs were similar to those at the wt P2X7, argues against a major effect of the truncated subunits.Thus, these truncated subunits do not or very inefficiently form functional homotrimers and their contribution to the current can be largely excluded. However, they might form functional heterotrimers with ANAP-labeled full-length subunits and as such, could still contribute to the fluorescence signal. Nevertheless, the fact that the ECThese data have now been included as Figure 5—figure supplement 2.It seems that calcium induces fluctuations of ANAP fluorescence. Does calcium have any direct effect on ANAP fluorescence?2+ and found that Ca2+ increases ANAP fluorescence, but only at concentrations above 100 mM. We included these photometric data in Figure 6—figure supplement 1.To test this possibility, we measured the emission spectra of ANAP-containing recording solution in the absence and presence of 10 μM, 100 μM, 1 mM, 10 mM, 100 mM, or 1 M Ca2+-induced fluctuations in VCF-measurements occurred also in the absence of ANAP .It needs to be considered, however, that the CaIt seems that fluorescence changes at some positions of the extracellular head domain and intracellular N-terminus track the process of facilitation. Tracking of this process is probably the most interesting finding of this paper, since the mechanism of facilitation is not known. In the paper, facilitation is measured as the increase of channel opening kinetics upon multiple applications of ATP. However, the first application of ATP clearly shows a small, fast component and a bigger, slow component . Could it be that the first component reflects initial gating and the second one the slow facilitation process that occurs upon prolonged ATP application? Could the fast kinetics of K217* fluorescence match the fast component of current, indicating that K217* tracks the initial ATP opening step but not current facilitation? Similarly, on Figure 6 it looks like TMRM kinetics match first the fast current component, while F11* fluorescence matches the kinetics of current facilitation. Could the authors comment on it?Indeed, current changes appear to be biphasic and we cannot exclude that the fast current component from K127* and TMRM-labeled F11*/S124C P2X7 match initial fast gating that occurs before the onset of facilitation and is reported by ANAP in position 127 or TMRM in position 124. However, the initial fast current component increases substantially between first and second ATP-application. This is not mirrored by the fluorescence signals, which are virtually identical between first and second ATP-applications. Therefore, we think that the fluorescence for this mutant reports a different process. Also, taking into account the proximity of the fluorophores to the orthosteric binding-site we consider it more likely, that these mutants report a process related to ligand binding. Compare also new Figure 3—figure supplement 3.Reviewer #2 (Recommendations for the authors):To strengthen the points of weakness above and to elevate the scientific merits of the paper, I have some comments which require attention.(1A) I am curious to know the P2X7 structural differences by VCF between just before the 1st application and just before the 2nd application of ATP, to clarify the structural background of "memory" of ATP application.Comparing the structures before first and second ATP application is very difficult with an indirect method like VCF, which provides mainly information about dynamic processes. Determination of different receptor states with this method would require clearly distinct fluorescence signals upon first and second activation, detailed structural information, and most likely numerous additional mutants to deduce and confirm specific conformational changes. Based on our observations, we generally suggest a potential transition into a pre-activated state (line 446) but do not want to elaborate on this to avoid overinterpretation.2+, i.e. Ca2+ influx via P2X7.(1B) Will there be any difference in (1A), in the presence and absence of extracellular Ca2+ (1 mM) were performed to test for Ca2+-specific effects  but revealed large unspecific signals (even in the absence of ANAP) in the spectral range of ANAP emission and thus hampered analysis. Recordings in 0.5 mM Ca2+ from oocytes expressing F11* and S124* did not reveal obvious changes in the pattern of ANAP fluorescence signals compared to measurements in divalent-free recording solution.Experiments in presence of extracellular Ca2+/CaM (Lines 335-342), However, there are no experimental results to support or deny them. It is desired to examine the effect of experimental manipulations of these factors on the F(and I) facilitation .(2A) Facilitation of F change was detected e.g. in S124*, F11*, S127C. Possible mechanisms of facilitation are discussed including mediators such as GDP, cholesterol, palmitoylation, CaWe performed additional experiments to investigate effects of palmitoylation  and a CaM interaction using a mutant with mutated CaM-binding motif and investigating FRET between ANAP-labeled P2X7 and mNeonGreen-tagged CaM . These data confirmed that deletion of a cysteine-rich intracellular region eliminates current facilitation  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. Please see Essential Revisions points 1and 2 for details.We would also like to point out that the changes observed in our study are compatible with a facilitation process that is an intrinsic property of the channel and does not require intracellular factors.(2B) Line 273: "the so-called current facilitation in P2X7 is due to a change in receptor gating rather than ligand binding", Line 366: "Ligand binding is unaltered between the 1st and 2nd activation and consequently, changes in channel gating account for the observed current facilitation". I wonder if this is correct, as F facilitation is observed in S124* which is close to the ATP binding site .As described, the fluorescence changes can report both ligand interactions and protein movements as well as combinations of both. The specific effects depend not only on the orientation and position of the mutated residue but also the fluorophore used . The fact that some mutants, like TMRM-labeled double mutant F11*/S124C and K127* detect fast processes (likely associated with direct ligand interactions or movements during ligand binding), does not exclude the possibility that fluorophores in neighboring positions (like ANAP-labeled S124*) detect relative domain movements within the receptor (likely resulting from a quenching/dequenching by aromatic residues or changes in the polarity of the environment) that contribute to the opening mechanism.(2C) F facilitation was detected not in S124C-TMRM , not in K127* , but in S124* . I cannot fully understand the ground of differences.ANAP can detect localized changes in its environment in a distance of about 5 Å. Therefore, the immediate environment can be completely different for S124* and K127* and ANAP can even report different changes in neighbouring positions. In case of ANAP-labelled S124* and TMRM-labeled S124C, the very differently sized fluorophores most likely sense different environments and therefore also report different interactions/effects (quenching or polarity).2+/CaM are discussed (lines 407-415). The authors showed limited structural rearrangements of the ballast domain upon ATP application, but there is no evidence by VCF to prove activation by intracellular ligand. I would like to see data of F change of such as A564* in the ballast domain in response to manipulation of intracellular factors.(3) As to the function of the ballast domain, it is described "intracellular ligand or interactors are required to "activate" this domain" (line 421). A possible involvement of the ballast domain to the functional modulation of P2X7 by the binding of various factors such as GTP/GDP, LPS and Ca2+. For details, please see Essential revision points 1 and 2. These data are now included in Figure 3—figure supplement 3, Figure, Figure 5—figure supplement 3, Figure 6—figure supplement 1, and Figure 6—figure supplement 3.In contrast to patch-clamp experiments, direct manipulation of intracellular factors during the recording is difficult if not impossible in TEVC experiments. As an alternative, we generated mutations (as suggested) in the palmitoylation site and the CaM binding site and performed FRET experiments with co-expressed ANAP-labeled P2X7 and mNeonGreen-tagged CaM. We further did experiments in different concentrations of extracellular Ca(4) Figure 4C: Does the F facilitation in T340* truly reflect "facilitation", in spite of the absence of current facilitation (as described in line 376)? The authors explained that a lack of faster current in the 2nd application is due to "strongly facilitated gating" (line 377). If that is the case, why there is F facilitation?The beauty of VCF is the ability to differentiate between electrophysiologically defined receptor states (open and closed) and associated domain movements. It is well possible, that due to a "leaky" gate "naive" and facilitated receptor states cannot be resolved electrophysiologically, while the associated domain movements can still be detected. In principle, the mechanism of the hinge (gate movement measured by fluorescence) does not change when the door  has a hole.2+?5) Do the F change of R-Geco  and FRET change by mNeonGreen  disappear in the absence of extracellular Ca2+-sensor and fluorescence changes were not expected in the absence of extracellular Ca2+. Since the FRET approach was discontinued, these control experiments were not performed.R-GECO is a widely used CaReviewer #3 (Recommendations for the authors):1. Please cite this paper when talking about eRF-E55D as well, https://doi.org/10.7554/eLife.37248.Thank you, done (line 103).2. The 3'-terminal CCA-sequence is already in the pANAP plasmid. More elaboration on how it augmented the translation is needed to make it clear to readers.X. laevis oocyte-endogenous tRNA-nucleotidyltransferases. However, we decided to add it, to provide a functional tRNA ready for aminoacylation by the ANAP-specific tRNA synthetase already upon cytoplasmic injection. To avoid misunderstanding, we replaced "augmented" by "provided".The 3’-terminal CCA sequence is important for aminoacylation and translation. The pANAP sequence described in  in the Supporting Information S2 encoding the tRNA lacks a 3’-CCA. This sequence might be dispensable, as it would likely be added by 3. Additional FRET experiments might be out of the scope of this paper, but please add more discussion and references regarding this possibility in the paper.We included several references to illustrate the potential of ANAP as a FRET partner more clearly (lines 328ff).4. One area that could be improved on is the uncoupled "ballast" domain. How might GTP/GDP and dinuclear Zn2+-binding rearrange this domain? Can pharmacological manipulations be used to change intracellular GTP/GDP or Zn2+ levels to see how Anap labeled in this domain changes its fluorescence differently? Could the palmitoylation of the anchor domain play a role for uncoupling the "ballast' domain and for the current facilitation?2+ levels in a time scale that is amenable to our VCF analysis. As described above, we analyzed the Cys-free mutants that are likely not palmitoylated. These analyses confirmed the importance of palmitoylation for the facilitation process and are now included . However, expression of these mutants was too low for VCF analysis and no obvious effects of palmitoylation on fluorescence amplitude or kinetics could be detected. This is now mentioned in lines 190-205.We are not aware of any protocols to change intracellular GTP or Zn5. Line 634, should be "artifacts"?Thank you, corrected!


Protein–proteininteractions (PPIs) form complex networksto drive cellular signaling and cellular functions. Precise modulationof a target PPI helps explain the role of the PPI in cellular eventsand possesses therapeutic potential. For example, valosin-containingprotein (VCP/p97) is a hub protein that interacts with more than 30adaptor proteins involved in various cellular functions. However,the role of each p97 PPI during the relevant cellular event is underexplored.The development of small-molecule PPI modulators remains challengingdue to a lack of grooves and pockets in the relatively large PPI interfaceand the fact that a common binding groove in p97 binds to multipleadaptors. Here, we report an antibody fragment-based modulator forthe PPI between p97 and its adaptor protein NSFL1C (p47). We engineeredthese antibody modulators by phage display against the p97-interactingdomain of p47 and minimizing binding to other p97 adaptors. The selectedantibody fragment modulators specifically disrupt the intracellularp97/p47 interaction. The potential of this antibody platform to developPPI inhibitors in therapeutic applications was demonstrated throughthe inhibition of Golgi reassembly, which requires the p97/p47 interaction.This study presents a unique approach to modulate specific intracellularPPIs using engineered antibody fragments, demonstrating a method todissect the function of a PPI within a convoluted PPI network. Misregulation of these cooperativePPIs has been shown to cause diseases such as cancer and neurodegeneration.4 Aberrant PPIs include either the loss of a crucial interaction orthe gain of a spatiotemporally incorrect interaction.5 The advancement of proteomics has facilitated the understandingof PPI networks, specifically in elucidating the interacting proteinpartners.7 Precisely mapped PPI networks provide fundamentalinformation to explore the possibilities in controlling cellular functionsthrough specific modulation of protein complexes. Therefore, we seeksystematic methodologies for PPI inhibition or stabilization to understandthe function of PPI and select the targets with relevant therapeuticavenues for disease treatment.9Protein–proteininteractions (PPIs) are essential for intracellularsignal transduction and transcriptional regulation.10 orknockout assays11 do not offer such PPI-specificregulation as the depletion of one protein will remove all of itsPPIs simultaneously. Likewise, overexpression of the protein of interestcan lead to multiple enhanced PPIs, cumulatively affecting the network.Therefore, the development of PPI-specific modulation can benefitfrom selective blockade of the PPI interface. Extracellular proteinsare readily orthosterically inhibited by antibodies, whereas smallmolecules and peptides are most often targeted to intracellular PPIs.However, a lack of pockets or grooves and the relatively large areaof the PPI interfaces pose challenges for discovering small-moleculemodulators, making it difficult to investigate multiple related PPIsrapidly and systematically.14An ideal PPI-specificmodulator tool should focus on the interactionbetween the protein partners of interest, leaving other functionsand PPIs of the targeted protein partners unaltered. Efficient geneticmodulation methods such as knockdown16 precisely refining the intracellular function of these antibodiesto the targeted cellular milieu. The variable platforms of antibodiesare available from nanobodies (∼15 kDa),17 single-chain variable fragments ,18 and antigen-binding fragments 19 to full-length IgG (∼150kDa), offering a tunable size range to tackle different PPI interfaces.Moreover, the constant chain within these antibody platforms enhancestheir stability against hydrolysis.21 Nevertheless,antibody-based modulators so far are mainly applied to secreted proteinsor cell membrane targets.23Antibodies possess unique properties as potential intracellularPPI regulators when compared to small molecules and peptides. Theprotein nature of antibodies allows convenient cloning modificationsto install subcellular localization signals,26 Dissecting the particularfunction of an individual p97/adaptor protein interaction is thereforeimportant but complicated within the p97 PPI network. In this work,we engineered antibody fragment inhibitors via phage display to modulatethe interaction between p97 and its adaptor protein, NSFL1C (p47)interacts with more than 30 adaptor proteins to regulate multiplecellular functions, including the maintenance of protein homeostasisand facilitating protein degradation.1C (p47)1. We cho29 Furthermore,antibodies against p97 would likely result in the inhibition of multiplePPIs because most adaptors bind to a common site on the p97-N domain;13 different p97 adaptors contain a UBX domain.31 Thus, inhibition from the p47 side should be more specific. To engineerantibodies for the p47-UBX domain, we carried out phage display usinga previously developed Fab phage library.32 The Fab scaffold derived from the 4D5 anti-HER2 antibody is highlystable (Tm 80 °C) and designed with amino acid variationsin four of the six complementarity-determining regions (CDRs).33 After four rounds of selection against the p47-UBX domain, morethan five clones were identified through phage ELISA (S1). These selected Fab hits (We chosethe UBX (ubiquitin regulatory X) domain of p47 as the antigen to discoverinhibitors of p47/p97 by Fab phage display. Previous studies haveshown that the p97-N terminal domain directly interacts with the p47-UBXdomain, and we hypothesized that some anti-p47-UBX antibodies wouldinhibit the p47/p97 interaction.ge ELISA 2a and S1Fab hits 2b for p4S2). We also evaluatedthe selectivity of these scFvs against the p97-binding domain of otheradaptor proteins  using BLI. All five scFv clones were highly selectivetoward the p47-UBX domain when compared to the NPL4-UBXL and FAF1-UBXdomain (S3). Moreover, as p47 and p37 haveoverlapping functions and are highly homologous ,34 it wascritical to ensure that the selected anti-p47-UBX scFv did not bindto the p37-UBX domain. Upon evaluating 2 anti-p47-UBX scFv nanomolarbinders with BLI, scFv-E04 did bind to p37-UBX with a dissociationconstant (KD) of ∼4 nM, while scFv-A06did not bind to p37-UBX . Overall,scFv-A06 is the most selective clone for the p47-UBX domain with a KD of ∼3 nM.Next, we assessed the bindingaffinity between the anti-p47-UBXscFv and p47-UBX domain using biolayer interferometry (BLI). Afteranchoring biotinylated p47-UBX on the BLI sensor tip, an increasingamount of each scFv was separately incubated with the p47-UBX-decoratedsensor. From the dose response profiles, scFv-A06 and scFv-E04 demonstratednanomolar binding affinities to the p47-UBX domain disrupted the p97/p47-UBX interaction, regardless of the presenceof nucleotide (S6). In the presence ofan equal molar amount of p47-UBX and scFv-A06 (both at 50 nM), thebinding of p47-UBX to the p97-anchored sensor was reduced ∼50%when compared with the control group where scFv-A06 was not present.Recent data indicated that there are three p97-binding modules onp47.38 Our construct for phage displayincluded the C-terminal SHP motif and UBX domain but lacked the N-terminalSHP motif of p47 . We thereforeevaluated the full-length p47 and found that the same scFvs that inhibitedthe p97/p47-UBX interaction also blocked the p97/full-length p47 interaction, demonstrating that our p47construct for phage display was adequate for selecting inhibitorsof the full-length p47.Althoughselected based on binding tothe p47-UBX domain, an essential requirement for these anti-p47-UBXantibody fragments is that they can disrupt the PPI of interest. Weemployed surface plasmon resonance (SPR) to test if these scFvs interferedwith the p97/p47 interaction. As a flow-based approach, SPR offersa dynamic monitoring of the PPI status.39 scFab (scFab-NLS) format in order to vary their cellularlocalization. Each clone contained a C-terminal HA (human influenzahemagglutinin) epitope tag for detection purposes. After transfectingthese plasmids with Xfect transfection reagents for 24 h in humanbone osteosarcoma epithelial (U2OS) cells , we visualized the antibody fragments and p47 by immunofluorescence(IF) ,41 scFab (∼60 kDa) primarily localized in the cytosol, whereasscFab-NLS was mostly confined to the nucleus and overlapped with p47.Due to the low intensity of cytoplasmic p47, it was difficult to confidentlystate that scFab-A06 colocalized with p47 in the cytosol. Taken together,IF results indicated that the molecular weight of the antibody fragmentsand the nuclear localization tag determined the localization of theantibody fragment. Western blotting of the scFab and scFv antibodyfragments indicated that E04 was expressed less than A06 antibodyfragments in U2OS cells, possibly due to a lack of stability .Translating these antibodies into functionalPPI modulators requires their intracellular presence. Therefore, wecloned the encoding sequences of these antibody fragments into a mammalianexpression vector. In the mammalian expression vector design, we includedboth the scFv and scFab (single-chain Fab fragment) formats and theN-terminal nuclear localization signal (NLS)-taggedS) cells S8, we viFigure S10). This result contrasted withour biophysical experiments, where these antibody fragments couldcompletely compete with full-length p97 . The cellular result was understandable for a few reasons. First,endogenous levels of protein expression were not precisely controlled,in contrast to our biophysical assays using recombinant proteins;hence, the expression level of scFv-A06 when compared to that of p47might have been insufficient to completely displace p47 from p97.Second, as previous studies have shown that p47 has an oligomericform,42 one p47 “arm” mighthave been inhibited by antibody fragments while the other(s) remainedbound. Third, a recent report demonstrated that the UBX domain isnot the only module of p47 that binds to p97.38 Our antibody fragments might have blocked only the UBX domain and/orthe C-terminal SHP motif of p47 that were present in the selectionconstruct  during phage display,while the N-terminal SHP motif of p47 retained its interaction withthe adjacent p97 protomer.We also confirmed the p47/antibody fragment interaction throughco-immunoprecipitation (co-IP) of p47. All A06- and E04-based antibodyfragments  bound to p47 3b. Bindi43 to quantitatively probe the effectof engineered antibody fragments on the p97/p47 interaction. The optimizedNanoBRET assay for p97/p47 PPI contained an N-terminal nanoluciferase-taggedp97-N domain (pNLF1N-p97-N) as the donor and a C-terminal Halo-taggedp47-UBX domain (pHTC-p47-UBX) as the acceptor . During the NanoBRET evaluation, the donor, acceptor,and antibody fragment (scFv and scFab)-expressing plasmids were co-transfectedinto the p47-KO U2OS cells to evaluate the proximity change betweenthe NanoBRET pair . In parallel,we employed the scFv and scFab platforms of the anti-RNase (ribonucleaseA) antibody44 as negative controls andthe p47-expressing vector (p47-FLAG) as the positive control. Whencompared to the BRET ratio in the scFv-RNase and scFab-RNase controlgroups, the co-transfection of either scFv-A06 or p47-FLAG similarlyreduced the BRET ratio by ∼15%, indicating a reduced interactionbetween the p97/p47 NanoBRET pair (S12c). Incomparison, the lack of effect on the BRET signal from scFv-E04 maybe attributed to its low expression level (We next utilized NanoBRET (bioluminescenceresonance energy transfer)assayacceptor 3c. To ruacceptor S11. Duriacceptor S11. Duri28 Therefore, we evaluatedthe effect of A06 and E04 anti-p47-UBX antibody fragments on Golgireassembly processes using the morphological distribution of GRASP55,a Golgi reassembly stacking protein that reflects the Golgi structure.45 After transfecting the antibody fragment-expressingplasmids into HeLa cells, the two scFv and scFab clones significantlyincreased Golgi fragmentation, with the scFv groups showing more Golgifragments per cell than the scFab groups (S13). Since the Golgi area per cell remained constant . The dual-inhibitionmechanism of E04 may have accounted for the effective inhibition ofGolgi reassembly, even given the lower expression level of E04 comparedto that of A06.Antibody fragment-basedPPI inhibitors are expected to affect thePPI-related phenotypes and modulate cellular functions. The p97/p47complex facilitates the fusion of Golgi membrane fragments to reassemblethe Golgi apparatus during the cell cycle at the late mitosis andearly interphase.constant 4d, an in28 Itwas therefore unexpected that expression of both the scFvs and scFabswithout an NLS tag yielded fragmented Golgi, while the scFabs withNLS tags showed minimal interference on the Golgi morphology. Thecytoplasmic distribution of antibody fragments was therefore correlatedwith activity, suggesting the importance of p47 during phases of thecell cycle where the nuclear membrane was intact.The tunability of the antibodyfragment platform contributed tothe understanding of p97/p47 PPI in Golgi reassembly. Previous mechanisticstudies revealed that the p97/p47 complexes participated in membranefusion during mitosis, whereas p47 primarily resided in the nucleusduring the interphase in HeLa cells.47 using purified rat liver Golgi (RLG) membranes.Our engineered scFvs for human p47 also bound to rat p47 . In the ex vivo assay,RLG membranes were treated with mitotic cytosolic lysates of HeLacells to form mitotic Golgi fragments (MGFs), that is, disassemblingthe Golgi cisternae into smaller vesicles (46 When scFv-A06 or scFv-E04was spiked into the interphase cytosolic lysates, the cisternal regrowthwas slowed down to less than 50% (To furthervalidate the effect of these scFvs on Golgi reassembly,we carried out a cell-free Golgi reassembly assay rat p47 S14. In t rat p47 S14. In t rat p47 S14. In t49 this antibody fragment inhibitor could potentially be useful inaiding the development of disease treatment.We have described the engineering of antibody fragment inhibitorsfor a specific PPI of the multifunctional protein p97 to modulatea cellular function of interest. These antibody fragments were selectedby Fab phage display for binding to the p97-interacting domain ofthe adaptor protein p47. Compared to genetic knockdown and knockoutapproaches, the well-defined steric blockade by these antibody inhibitorspotentially reduces the interference to the overall p97 PPI network.Such antibody fragments are available in tunable molecular weightsand subcellular localization tags through protein engineering, makingit an attractive approach to study the PPI of interest at differentcellular locations and providing toolkits to tackle biological questions.For example, this work demonstrates the first reported antibody fragmentthat modulates the Golgi structure. As Golgi defects are observedin an increasing number of human diseases,51 it is still challenging to develop inhibitorsfor highly related targets systematically. By contrast, phage-selectedantibody fragments can readily be developed for a panel of PPIs witha common hub protein. Furthermore, antibody discovery by phage displaycan also be applied to protein targets with post-translational modifications,protein isoforms, splice variants, or conformational variants, tremendouslyimproving the applicability of our antibody inhibitor platform. Currently,the cell membrane and endosomal entrapment of proteins limit the abilityto deliver antibodies intracellularly.52 Intracellular antibodies have been used as PPI modulators in a fewcases, such as the signaling pathway of RAS proteins, and we now showtheir utility for selectively blocking one PPI of a hub protein thatbinds to many proteins at the same site.55 Developmentof efficient and robust methods for delivering recombinant antibodies56 to intracellular PPI targets will further extendthe applicability of the technology for biomedical applications, ideallyby perturbing the formation of malfunctional protein complexes thatare associated with diseases and disorders.Although we andothers have worked to develop small-molecule inhibitorsof PPI,


In this work, three pairs of constitutionally isomeric imine-linked donor-acceptor (D-A) COFs are synthesized, which are different in the orientation of the imine bonds (D-C=N-A (DCNA) and D-N=C-A (DNCA)). The constitutional isomers show substantial differences in their photophysical properties and consequently in their photocatalytic performance. Indeed, all DCNA COFs show enhanced photocatalytic H2 evolution performance than the corresponding DNCA COFs. Besides the imine COFs shown here, it can be concluded that the proposed concept of constitutional isomerism of linkages in COFs is quite universal and should be considered when designing and tuning the properties of COFs.When new covalent organic frameworks (COFs) are designed, the main efforts are typically focused on selecting specific building blocks with certain geometries and properties to control the structure and function of the final COFs. The nature of the linkage  between these building blocks naturally also defines their properties. However, besides the linkage type, the orientation,  Systematic investigation of isomerism in covalent organic frameworks (COFs) can provide key insights into their properties. Here, the authors reveal that the constitutional isomerism of the linkage i.e., linkage orientations distinctly impact COFs’ structural and photophysical properties. Functionality and structural diversity are mostly introduced into such COFs by applying different linkers so far9. While there are intensive studies on linker chemistry, the COF linkages. i.e., the bonds formed between the linkers during crystallization, have gained much less attention even though they play a critical role in both for the design and the properties of COFs12. Currently, research on COF linkages is focused on exploring new linkages to increase the amount of available synthetic methods for reticulating organic building blocks but also for tuning the properties of the resulting COFs17. Strategies for post-synthetic modification of existing linkages, such as protonation19 or transformation to more stable covalent bonds23 were developed for the same purpose. Furthermore, synthetic methodologies to introduce mixed linkages such as boronate/boroxine and imine linkages into COFs have been described25. However, another obvious structural variation of currently available linkages, namely their orientation towards the linkers, has so far not been exploited in depth. Imine-linked COFs have been reported frequently due to the ease of their synthesis and relatively high chemical stability10. Such C=N-linked COFs are prepared by condensation of aldehyde- and amino-functionalized monomers, while there seems to be no rationale behind the choice, of which of the monomers should be aldehyde or amino-functionalized, respectively. Thus, presumably, just the cheaper or easier-to-prepare monomers are commonly chosen. However, this decision might significantly impact the properties of the final materials, as it predetermines the orientation of the C=N-linkage towards the different linkers yielding the possibility of constitutional  isomerism within this connection  are porous crystalline materials composed of organic building units (linkers) connected through strong covalent bondsion Fig. 26–28. So8, in which the interaction between the D and A moieties are crucial for the final properties and performance30. Indeed, Ghosh et al. observed at first no significant difference in the optical properties of two amorphous microporous polymer networks in which anthracene and pyrene moieties (thus two donors) were connected by imine bonds in reverse orientation. However, after doping with iodine, thus oxidation of the pyrene moiety, the crucial influence of imine linkage orientation on the electronic properties of the networks was revealed31.The constitutional isomerism of the linkages might be especially important for donor–acceptor (D-A) type COFs, currently vastly explored for different optoelectronic or photocatalytic applications18. In this work, we show that this is also valid for the pairs of constitutionally isomeric COFs.In imine-linked D-A COFs, two isomeric structures can be imagined, one in which the donor is linked to the imine carbon (D-C=N-A) the other where it is linked to the imine nitrogen (D–N=C–A) Fig. . In this32 whereas, triazine is an electron-accepting building block17. Finally, the triphenylbenzene monomers can serve as both donor and acceptor depending on the counter building blocks18. The three pairs of isomeric COFs were named DCNA-X and DNCA-X (X = 1–3), in which D stands for donor, A for acceptor, while CN or NC shows how the imine linkage is connected to the D and A nodes  topology. In addition, the intense and sharp diffraction peaks indicated their high crystallinity. The chemical structure of DCNA and DNCA COFs was further investigated by Fourier transform infrared spectroscopy (FTIR)  solid-state NMR  and DNCA-1 (1620 cm−1), showing that the imine linkages have a different chemical environment. On the other hand, the characteristic peaks of the triazine 34 appeared at almost identical wavenumbers  sorption. DCNA-1 showed a Brunauer–Emmett–Teller (BET) surface area of 1066 m2 g−1, whereas DNCA-1 showed a BET surface area of 456 m2 g−1, pointing to a slightly less ordering in the latter. Also, DCNA-2 shows a higher surface area than DNCA-2, while the opposite is seen for the DCNA/DNCA-3 pair  have been reported already and are synthetically accessible under mild solvothermal conditions DNCA-1 120 cm−1, μ, where Φ and Σμ correspond to the photocarrier generation yield and the total charge carrier mobility (combined electron and hole mobility), respectively35. Maximum photoconductivities of 4.0 × 10−6 and 2.0 ×10−6 cm2 V−1 s−1 were observed for DCNA-1 and DNCA-1, respectively, revealing higher photoconductivity of DCNA-1 compared to DNCA-1  to the CB. Upon light irradiation, DNCA-1 has a higher EPR signal intensity. The higher EPR intensity can also be attributed to the relatively better light absorption ability of DNCA-1. However, DNCA-1 showed just a ~33% increased light absorption  compared to DCNA-1  was used as acid for protonation, which also acts as the sacrificial reductant in the photocatalytic hydrogen production experiments shown later. AC protonated COFs were prepared via a 5-minute treatment in 0.1 mol/L AC aqueous solution followed by filtration and drying overnight by ultra-high vacuum at room temperature. The protonation of imine moieties was further confirmed by FTIR measurements, from which the imine peaks at ~1620 cm−1 disappeared, and new bands appeared at ~1790 cm−1  and in the presence of 0.1 M AC  as the sacrificial electron donor (SED) and Pt as co-catalyst. As expected, an immediate and obvious darkening of the color of the pristine COFs was observed when they were dispersed in the reaction solutions, indicating that the actual catalysts for photocatalysis are the protonated COFs   as SED in which the COFs retained their pristine chemical structures  at 420 nm of DCNA-1_AC was measured to be 1.49 %, while the AQE of DNCA-1_AC was too low to be accurately measured.To investigate the impact of the changing optoelectronic properties of constitutionally isomeric COF pairs on their photocatalytic performance, photocatalytic hydrogen evolution reaction (HER) from water with all COFs as photocatalysts was investigatedOFs Fig. . As expeOFs Fig. . DCNA-1_h−1 Fig. . The samh−1 Fig. . DCNA-2_43. The CB value of DCNA-1_AC decreased by 0.64 eV (∆CB) upon protonation, but its VB value is almost identical to the pristine COF DCNA-1. Similarly, DNCA-1_AC shows a decrease of CB position by 0.40 eV, and its VB position increased slightly by 0.1 eV (∆VB). Thus, protonation significantly decreased the CB positions of the COFs but did not change their VB positions much, which was also observed for the other two DCNA/DNCA pairs and their protonated counterparts  and finally enables the fast photocatalytic HER. In addition, the greater charge carrier mobility of DCNA-1_AC compared to DNCA-1_AC should facilitate the charge transfer to the metal nanoparticles as active sites for proton reduction, thus contributing to the better activity of DCNA-1_AC as well  light absorption and exciton generation, (2) charge carrier separation, transport, and recombination, and finally (3) surface reaction. Based on the optoelectronic properties observed before, DNCA-1_AC possesses broader light absorption (Step 1) and better charge separation efficiency (Step 2), which both should contribute to better photocatalytic performance. However, the photocatalytic results contradicted these observations. This led us to focus on the surface reaction step (Step 3) of the photocatalysis process. Valence band XPS (VB-XPS) was applied to detect the VB positions of all COFs before and after protonation  calculations were performed on the isomeric COFs DCNA-1/DNCA-1 and their protonated forms DCNA-1_AC/DNCA-1_AC Fig. . Using p1, hydrazone15, imide44, amide20, cyanovinylene45, amine21, oxazole22, thiazole46, or imidazole47 linkages , can be achieved for the strongest donor–acceptor combination. Experimental results and DFT simulation both point to the crucial influence of imine linkage orientation on bandgap and especially CBM to explain the different catalytic performances. This work shows that constitutional isomerism is a fundamental aspect to understand COF properties and performances. It is plausible that this concept can also be extended to various other linkages in COFs.In conclusion, we propose that the constitutional isomerism of the linkages, i.e., the orientation of chemical connections towards the COF building blocks, should be considered and treated with the same importance as the nature of the backbone functionalities when designing COFs. The imine linkage, as the most reported linkage in COF chemistry so far, was chosen as a representative to elucidate the effect of linkage isomerism in donor–acceptor COFs. Two kinds of constitutionally isomeric COFs were synthesized, namely DCNA (Donor-C = N-Acceptor) and DNCA (Donor-N = C-Acceptor) COFs with identical functionalities but opposite orientations of the imine linkage against the donor and acceptor motif. The linkage isomerism has a distinct impact on the photophysical properties of the constitutionally isomeric and isoreticular COFs. Consequently, COFs with different linkage orientations show distinct differences in their performance as photocatalysts for the HER from water. HER rates as high as 83.66 μmol h2 bath) and degassed by three times freeze-pump-thaw cycles. The internal pressure was evacuated to 10−3 mbar. Then, the tube was sealed and heated at 120 °C for 3 days. The final fluffy precipitate was filtered and washed with acetone several times. Finally, the COF powders were dried in a normal oven at 80 °C. The specific combinations of the solvents are given in Supplementary Note Generally, all the COFs were synthesized by the solvothermal method in a Pyrex tube at 120 °C for 3 days. A Pyrex glass tube (15 mL) was charged with the donor and acceptor monomers, specific solvents, and 6 M acetic acid aqueous solution. The tube was first sonicated until a fluffy solid formed and then flash frozen at 77 K (liquid N2PtCl6 aqueous solution (8 wt%) were added as the source of Pt co-catalyst. The reactor was sealed with rubber stoppers and degasses by Argon for 30 minutes before irradiation. Then, the reactor was irradiated with a 300 W Xe lamp (L.O.T-Quantum design) with appropriate filters. The temperature of the system was kept at 20 °C by water circulation. The gas sample was taken from the headspace, and H2 was quantified by GC (Agilent 7820 A) equipped with a thermal conductivity detector. The photocatalytic hydrogen evolution reaction rates in AC were determined from the linear regression fit of the H2 evolution curves, and the pressure increase was neglected in the calculations. Due to the negligible amount of H2 evolved by DNCA/DNCA-X_AC COFs, H2 was only measured after finishing the irradiation. All the reacted COFs were recovered by filtration, washed with acetone, and dried at 80 °C overnight for further characterization.The photocatalysis was conducted in a 36 mL side irradiation quartz reactor equipped with a 152 mL glass gas container. Generally, 3 mg pristine COF were dispersed in either 16 mL 0.1 M AC aqueous or triethanolamine (TEOA)/water (v:v = 2 mL: 16 mL) solution. 3 microliter H49, version 5.4.4. Atomic positions and cell parameters were optimized using the PBE functional50, while band structures were calculated using HSE52. The D3 dispersion correction53 with Becke-Johnson damping was used54. The kinetic energy cutoff was set to 600 eV, and a 2×2×1 k point grid was used. The counterion for calculations on the protonated forms was ascorbate. The cell vector perpendicular to the layer was set to 20 Å, resulting in a vacuum space of at least 12 Å for the protonated COFs. Elementary cells are shown in Supplementary Fig.Calculations on periodic models were performed with VASP55, version 4.0.0.2. The cluster model was cut out of the periodic structures and contained one whole pore . Only the protonated imine groups were optimized, while all other atoms were kept fixed. This optimization was done with PBE functional50 and with the D3 dispersion correction53 with Becke–Johnson damping54, using the 6-311G** basis set56. CAM-B3LYP60 calculations were performed using the def2-SVP basis set61, which gives very similar results compared to the larger 6-311G** basis set. Time-dependent density functional theory (TD-DFT)62 was used to calculate excitation energies and oscillator strengths. Natural transition orbitals (NTOs)63 were used to analyze the electron-hole pairs contributing to the excitations.Cluster calculations were performed using ORCASupplementary Information