Distinct activation mechanisms of β-arrestin-1 revealed by 19F NMR spectroscopy

β-Arrestins (βarrs) are functionally versatile proteins that play critical roles in the G-protein-coupled receptor (GPCR) signaling pathways. While it is well established that the phosphorylated receptor tail plays a central role in βarr activation, emerging evidence highlights the contribution from membrane lipids. However, detailed molecular mechanisms of βarr activation by different binding partners remain elusive. In this work, we present a comprehensive study of the structural changes in critical regions of βarr1 during activation using 19F NMR spectroscopy. We show that phosphopeptides derived from different classes of GPCRs display different βarr1 activation abilities, whereas binding of the membrane phosphoinositide PIP2 stabilizes a distinct partially activated conformational state. Our results further unveil a sparsely-populated activation intermediate as well as complex cross-talks between different binding partners, implying a highly multifaceted conformational energy landscape of βarr1 that can be intricately modulated during signaling.

1.The authors interpret all 19F spectral changes as funcfionally relevant structural changes.However, the introduced PSP-6F label has a considerable size, has a rigid pyridine ring, and may make a number of arfificial interacfions with the surrounding groups.Furthermore, the appearance of one or several 19F lines may be a subtle effect depending on the exact chemical shift differences and the exchanges rates between different conformafions.There is no caufion about this whatsoever in the manuscript.Instead, all the spectral changes are interpreted at face value as important structural changes.Since there is no unique relafion between chemical shift and structure -and this is parficularly true for the double CF3labeled pyridine -any observafion of chemical shift changes can only be idenfified with a relevant structural change if the observafion is corroborated by orthogonal evidence.The current manuscript does not meet this requirement.
2. The derived findings on the acfivafion of βarr1 are not new and give no new mechanisfic insights.Basically, all conclusions have already been described in the literature: (i) Figure 2: it is well known that V2Rpp must displace the β20 strand to form an anfiparallel β-sheet with the β1 strand (see Shukla, Lefkowitz, Kobilka and others).Changes in the N-terminal region upon V2Rpp are therefore expected.The appearance of two lines for the T6C mutant is interesfing, but it may well be an effect of the changed side chain.This is not followed up.
(ii) Figure 3: the disrupfion of the polar core by V2Rpp binding is also very well known.Hence perturbafions in this region are not surprising.
(iii) Figure 4: similarly, the 'crest' or hinge region between N-and C-domains is known to undergo structural changes upon V2Rpp binding.Here mulfiple lines are observed for the L71C mutant.However, this is a single observafion and again it is unclear whether the side chain subsfitufion does not introduce arfifacts or whether the appearance of several lines is due to a parficular rafio of exchange rates and chemical shift differences.
(iv) Figure 5+6: the effect of PIP2 binding on the hinge region between N-and C-domain has recently been described in detail by Janetzko et al. (2022) using FRET and fluorescence reporters on the finger and gate loops.Again, structural changes of the residues shown as perturbed in Figure 6b are completely expected.The quesfion remains unanswered how the signal is transduced from the PIP2 binding site to e.g.E313.
Further points: 1.The exact construct for full-length βarr1 should be given as a sequence, including the starfing and end residue numbers.2. Figure 1b: the V2Rpp bound βarr1 looks like ΔCT. 3. Figure 2d: the T6C heterogeneity should be followed by fitrafion.The used V2Rpp concentrafion may not be sufficient to displace β20 completely. 5.The affinity for V2Rpp should be derived from the data in Figure S6a. 5. Figure 3: the interpretafion of the NMR peaks as corresponding to the various gate loop conformafions is highly speculafive.6. Figure 6b: the colors of perturbed vs unperturbed residues are very hard to disfinguish.7. The chemical characterizafion, synthesis and supplier of the PSP-6F label are not indicated.The given reference #30 does not provide any clues.

Reviewer #2 (Remarks to the Author):
Via cysteine mutagenesis, the authors use 19F NMR to invesfigate conformafional response of both a CTtruncated and full-length version of Beta-arresfin-1, as a funcfion of various phospho-pepfides, V2Rpp, and PIP2.The work builds upon a recent study by Janetzko et al, but with a focus on conformafional ensembles.I think the work is interesfing and above the bar for publicafion in Nature Commun.It might have been extremely interesfing to aftempt NMR spectra in the presence of a receptor but I certainly appreciate how much more difficult this would be.While for the most part, PIP2 and V2Rpp seem to act independently, the modest enhancement of the acfive signature at D390 for example, speaks to the inherent cooperafivity and allostery present in the system.In tesfing the class A and class B receptor responses and the connecfion to transient and long lived desensifizafion, is it sufficient to use V2Rpp or phospho-pepfides from the Beta-2 adrenergic receptor or are there addifional differenfiators in the receptor itself?I am assuming that beta-Arr1 can undergo both transient and long-lived desensifizafion.In this case, it might be helpful to use something like alpha-fold to idenfify the class A and B complexes and point out interacfion surfaces on Barr-1, how they might differ, and how this might be reflected in the current data.Minor comments: 1. Abstract -" We further show that the membrane phosphoinosifide PIP2 independently modulates βarr1 conformafional dynamics without displacing its autoinhibitory carboxyl tail, leading to a disfinct parfially acfivated state."Unless I am mistaken, this was the conclusion of the Cell paper by Janetzko et al.This sentence should therefore be expressed as a validafion from this work rather than a new independent discovery.2. Fig. S2A." The data suggest that while all states show a certain degree of structural dynamics, the V2Rpp-bound state exhibit the highest heterogeneity with many sites displaying two or more conformafions (Supplemental data Fig.S2a).In parficular, residues in the central crest adopt a uniform conformafion in the basal state, while they show mulfiple conformafions when acfivated."Heterogeneity is harder than this to describe.Configurafional entropy for example (sum of all n states pn*ln(pn)) might be one way but this excluded heterogeneity of a given state which could in principle be a funcfion of the linewidth of each state (although we'd need to know the homogeneous and heterogeneous contribufions).The point is that heterogeneity is not well defined here.Perhaps the authors were talking about heterogeneity between different domains?This is also something befter answered by T2 relaxafion experiments.Either way, this should be more rigorous.3. Grammar.The paper is well wriften but there are some awkward or grammafically incorrect wording sentences: -abstract "evidences highlight" -page 3 "have urged for", "evidences", "phosphorylafion of [the] receptor" -page 4 "parfially acfivafion" -page 7 "site exhibit more" -page 11 "involvement in [the] Barr acfivafion pathway" -page 13 "the distal CT regions does modulate" -page 14 "the structural and dynamics changes" -page 15 "combinatory effects" page 18 "MestraNova" page 24 "states crystal structures" I have two main problems with this work (detailed below).For the given reasons, I recommend to publish the work in a more specialized journal after suitable revision.

RESPONSE
Main points: 1.The authors interpret all 19F spectral changes as functionally relevant structural changes.
However, the introduced PSP-6F label has a considerable size, has a rigid pyridine ring, and may make a number of artificial interactions with the surrounding groups.
Furthermore, the appearance of one or several 19F lines may be a subtle effect depending on the exact chemical shift differences and the exchanges rates between different conformations.
There is no caution about this whatsoever in the manuscript.
Instead, all the spectral changes are interpreted at face value as important structural changes.
Since there is no unique relation between chemical shift and structure -and this is particularly true for the double CF3-labeled pyridine -any observation of chemical shift changes can only be identified with a relevant structural change if the observation is corroborated by orthogonal evidence.The current manuscript does not meet this requirement.

Response:
We thank the reviewer very much for the critical comments and suggestions.We would like to address the reviewer's concerns from the following aspects.
1) First of all, we agree with the reviewer that exogenous probes introduced through cysteine mutations should be interpreted with caution.Actually, we chose the wPSP-6F probe because of its relatively smaller size together with several other advantages.The work describing the development of this probe has been published in Angew Chem Int Ed Engl recently (Chai et al, Angew Chem Int Ed Engl, 2023, 62, e202300318), and we have updated this reference in the revised manuscript (ref. 21).In the Figure 1 below, we show the chemical structures of wPSP-6F compared with another three different probes that have been used in studies of GPCR or βarrestin conformational changes, including the commonly used Cy3 for fluorescence study (e.g.Asher et al, Cell, 2020, 185:1661), the commonly used commercial 19 F probe BTFMA (e.g.Huang et al, Cell, 2021, 184:1884), and a trimethylsilyl group-based 19 F probe TMSiPhe (Liu et al, Nat. Commun., 2020, 11:4857).We can see that the wPSP-6F probe is shorter in its sidechain length and smaller in its overall size compared to the other probes.Its smaller size is less likely to introduce steric hinderance to local structure, and its shorter sidechain length enables higher sensitivity to local conformational changes.Apart from these, this probe has several other advantages (as described in ref 21), including mild requirements for ligation, high signal sensitivity (based on six equivalent fluorine atoms and fast trifluoromethyl rotation), as well as its high environmental sensitivity.Taken together, wPSP-6F is a preferred probe to meet the needs of our current study.2) To confirm that all 19 F-labeled samples maintain structural and functional similarity to the wild-type protein, we collected the 1 H NMR and CD spectra for all 19 F-labeled samples, and performed both Fab30 and clathrin binding assays.Briefly, the 1 H NMR and CD spectra for all samples are essentially similar to the wild-type protein, and all samples exhibit V2Rppenhanced Fab30 and clathrin binding activities.These results together verify that the 19 F-labeled βarr1 proteins retain the structural and functional characteristics of the wild-type protein.These data are shown in Supplementary Fig. 2-4 of the revised manuscript, and also attached below.(Supplementary Fig. 2 of the revised manuscript) (a) 1 H NMR spectra of the 19 F-labeled βarr1 samples compared with the wild-type full-length protein showing the amide signal region.The five 19 F-labeled samples shown at the bottom (D240C, C242, C269, H198C and L191C) were prepared using a different batch of buffer in which cocktail protease inhibitor, TFA and DTT were pre-added, and was therefore compared with a WT sample using the same buffer.The strong peaks in the 7.5-7.7 ppm region originate from the small molecules.(b) 1 H NMR spectra of the 19 F-labeled βarr1-ΔCT samples compared with the wild-type βarr1-ΔCT showing the amide signal region.3) To further address the reviewer's concern that the introduced probe "may make a number of artificial interactions with the surrounding groups", we performed molecular dynamics simulations for a number of representative labeling sites in different structural regions.The simulation results indicated that βarr1 structure remains stable and not perturbed by the labeling, and revealed no additional or artificial interactions of the wPSP-6F probe with nearby residues in all systems simulated.The MD results are shown in Supplementary Fig. 5 of the revised manuscript, and also attached below.No apparent artificial interactions between the probe side chain and nearby residues are observed during the simulations.4) To provide additional experimental support for the notion that the observed multiple peaks do not originate from artificial interactions of the wPSP-6F probe, we have also tried labeling with different 19 F probes for representative sites, such as L71C and T6C.As shown in the Supplementary Fig. 9 of the revised manuscript, the L71C mutant labeled with either BTFMA-6F or BTFMA also show spectral changes from an apparently symmetric single peak in the apo state into obvious co-existence of multiple peaks when V2Rpp is present.Spectra deconvolution suggest the presence of three or more peaks that overlap with each other.The overall spectral changes of these two probes with different chemical structures recapitulates the observation of wPSP-6F, suggesting that the multiple resonances detected in the activated state is not likely to be a probe-induced artifact.In the wPSP-6F labeled sample, the separation of the multiple resonances is clearer, also reflecting the relatively high environmental sensitivity of this probe as described in the published Angew paper (ref. 21).
Similarly, for the T6C site, dual resonances were also observed in the V2Rpp-bound state when the protein is labeled with different 19 F-probes.The corresponding results are shown in Supplementary Fig. 7 of the revised manuscript.
We also labeled V70C and G72C, two neighboring sites of L71C.V2Rpp binding also results in increased spectra heterogeneity for these two sites, showing overlapping resonances that deconvolutes into multiple peaks.Taken together, the multiple resonances most likely reflect conformational heterogeneity at this local area, rather than an artificial effect introduced by the wPSP-6F probe.
Below we attach the related panels from Supplementary Fig. 7 and 9 of the revised manuscript.5) In answer to the reviewer's concern about "the appearance of one or several 19 F lines may be a subtle effect depending on the exact chemical shift differences and the exchanges rates between different conformations", we made the following revisions: (a) We performed 19 F CEST experiments for a number of important sites that apparently show multiple peaks, and the CEST profiles help confirm that the two or more resonances do correspond to conformational states that are in exchange with each other.Based on the facts that the peaks can be separated in the 1D spectra, and that their exchange can be detected by the CEST experiment, these exchanges are estimated to occur in the upper millisecond time scales.The CEST data confirming exchange among different conformations are summarized in Supplementary Fig. 13 of the revised manuscript, and also attached here.(From Supplementary Fig. 6 of the revised manuscript) (c) We agree with the reviewer that the appearance or one or more NMR peaks depends on the exchange rates and chemical shift differences, and we are sorry that we did not make clear cautions about this in our former manuscript.In the revised manuscript, apart from more rigorous wording for the interpretation of the NMR results, we also added a brief caution about this point in the Discussion section (page 18, end of paragraph 1) as follows: "Here we would also like to note that the actual conformational landscape of βarr1 is expected to be more complex than the apparent NMR spectra suggest, because observation of a single peak not necessarily exclude the possible presence of more than one conformation due to limited spectral resolution, fast exchange between multiple conformations, or very low populations of additional conformations."6) Finally, we are aware that the usage of mutagenesis-based method to introduce exogenous 19 F probe would inevitably cause certain alterations to the local structure dynamics.Although the 19 F probe we used is much smaller than fluorescence probes and also smaller than other commonly used commercial 19 F probes, the labeled sample could not be exactly identical to the wild-type protein.Therefore, rather than looking at the absolute values of the spectral observables for each individual state, we focus on interpreting the changes or differences of the spectral behaviors for each site in different functional states (e.g.apo-state, ΔCT-state or in the presence of different binders).We argue that the relative changes of spectra can directly reflect the differential effects of different binding components on βarr1.In the revised manuscript, we paid special care to make this point clear when interpreting the data, and also added a brief discussion on this point in the Discussion section (page 16, first paragraph of the Discussion section) as follows: "Here we would like to caution that the method of introducing 19 F probe through mutagenesis would inevitably affect local structural dynamics.Therefore, the absolute values of the NMR observables for each individual state may deviate from the wild-type protein and should be interpreted with care.However, spectral changes for a giving site obtained in different functional states or in the presence of different binders could provide invaluable information, particularly regarding the different activating mechanisms induced by V2Rpp and PIP2." 2. The derived findings on the activation of βarr1 are not new and give no new mechanistic insights.Basically, all conclusions have already been described in the literature:

Response:
We appreciate the reviewer's critical and helpful comments.During the revision, we performed additional experiments to expand the scope of our study.
Firstly, we carried out 19 F CEST experiments, which verify and provide further information to the 1D experiments.In particular, the CEST results help reveal the existence of a low-populated intermediate conformation that could not be unambiguously identified from the 1D spectra.Therefore, we revised the manuscript and added a section " 19 F CEST data unveils an intermediate conformational state" to present the details of this observation (page 13-14 of the revised manuscript together with Fig. 5).
Secondly, we further investigated the potential cross-talks between the phosphopeptide and PIP2 binding events.Although being somewhat out of our expectation, the results demonstrated that the two pathways show rather complex interplay instead of being simply additive or cooperative.In the revised manuscript, a new section "Cross-talk between phosphopeptide and PIP2 binding" is added to describe the corresponding results (page 14-15 of the revised manuscript together with Fig. 6).
Thirdly, following the reviewer's comments, we performed further experiments to elucidate how PIP2 binding in the C-domain can be transferred to the back loop region.We demonstrate that PIP2 binding perturbs the packing in β15 and β16 strands, which can affect the neighboring C loop and back loop.We add these new results in the section "PIP2 induces βarr1 activation from the back side of the N-, C-domain interface" (page 11-13 of the revised manuscript, together with Fig. 4 and Supplementary Fig. 12).
Finally, we investigated the effects of membrane binding on βarr1 conformational dynamics using lipid nanodiscs.These results, together with the PIP2 data and the V2Rppinduced conformational changes in the C-edge loops, imply the existence of long-range allosteric effect between the N and C domains.We add these new results in a new section "Effects of membrane binding on βarr1 conformational dynamics" (page 15-16 of the revised manuscript together with Fig. 7).
Based on these new data and combined with our other results, we have re-written the Discussion section of the manuscript to summarize the new insights of βarr1 activation that are not readily obvious from previous literature.Here we would like to emphasize the main points of the new insights that can be obtained from our current work: (1) It is well-known that the conformations of βarrs as well as their interactions with receptors are highly dynamic (we have revised the introduction section to emphasize on this point, see page 3-4 of the revised manuscript).Available structures of βarr1 capture its inactive and active state conformations, however, the conformational dynamics are often quenched in the process of stabilizing these lowest-energy conformations.We argue that the aim and merit of our study is to provide a molecular mapping of the conformational dynamics changes of βarr1 during activation.The choice of a small 19 F probe (significantly smaller than fluorescent probes) offers the advantage of being able to monitor localized conformational fluctuations, and the labeling of over 20 sites provides a more complete structural coverage that allows us to obtain a detailed mapping of βarr1 conformational changes.
(2) The distinct patterns of spectral changes in different structural regions of βarr1 elicited by V2Rpp or PIP2 binding reveal that the conformational landscape of βarr1 is differentially modulated by the different binding partners.Although previous biochemical and fluorescence results (by Janetzko et al. 2022) have indicated that PIP2 binding induces an "active-like" conformation that can be reflected by fluorescence changes and Fab30 binding activities, etc., a detailed picture of whether this "active-like" conformation is actually the same as that induced by V2Rpp or CT release is lacking.Our current results demonstrate that the PIP2-induced conformational state, or conformational ensemble, is clearly distinct from that induced by CT removal or V2Rpp binding.
(3) The CEST data further reveal a hidden intermediate conformational state S* that is low populated and has not been previously identified.This S* state is different from either the V2Rpp-stablizied active conformation or the PIP2-induced "active-like" conformation, adding further complexity to the βarr1 energy landscape.The CEST and 1D NMR data together imply the existence of multiple "active-like" or "pre-activated" conformations that differ from each other in local conformational dynamics in different structural regions, underlying the multifunctionality of the βarrs.
(4) Our investigation of the cross-talks between phosphopeptide and PIP2 binding further reveals a complex interplay between the two factors.In some structural regions, the two binding events appear to have synergetic effects, while in other regions they show more complex behaviors and may sometimes be counteracting.These observations are also not reported previously.
Taken together, our current results demonstrate that PIP2 modulates βarr1 conformational landscape in a way different from phosphopeptide like V2Rpp, and suggest that apart from the well-accepted "phosphorylation barcode" mechanism by which phosphorylation patterns differentially modulate βarr conformations and functions, PIP2 binding and its interplay with the phosphopeptide can act as an additional layer of "barcode" that fine-tunes the βarr conformational landscape.
Below are brief responses to the individual points: (i) Figure 2: it is well known that V2Rpp must displace the β20 strand to form an antiparallel β-sheet with the β1 strand (see Shukla, Lefkowitz, Kobilka and others).Changes in the Nterminal region upon V2Rpp are therefore expected.The appearance of two lines for the T6C mutant is interesting, but it may well be an effect of the changed side chain.This is not followed up.

Response:
We agree with the reviewer that the V2Rpp induced changes at the TE region is expected.For the T6C site, we have added V2Rpp titration experiments as the reviewer suggested.The titration results indicate that 1.2-fold and 5-fold excess of V2Rpp show no spectral different.This result is included in Supplementary Fig. S7 of the revised manuscript and also attached below.We have also tested using different 19 F probes and obtained similar results (the observation of two resonances) as presented in our response above.(ii) Figure 3: the disruption of the polar core by V2Rpp binding is also very well known.Hence perturbations in this region are not surprising.

Response:
We agree with the comment.We have merged the previous Figure 3 with other data into a single figure (Fig. 2 of the revised manuscript) and shortened the corresponding section.
(iii) Figure 4: similarly, the 'crest' or hinge region between N-and C-domains is known to undergo structural changes upon V2Rpp binding.Here multiple lines are observed for the L71C mutant.However, this is a single observation and again it is unclear whether the side chain substitution does not introduce artifacts or whether the appearance of several lines is due to a particular ratio of exchange rates and chemical shift differences.

Response:
We thank the reviewer for the comment.We have merged the previous Figure 4 with other data into a single figure (Fig. 2 of the revised manuscript).For the L71C site, as mentioned in our response above, we tested using different 19 F probes as well as labeling at its two neighboring sites and reached similar conclusions.These results are shown in Supplementary Fig. 9 of the revised manuscript and also in the Fig. R7 above.
We have also run MD simulations of the L71C-labeled sample in the V2Rpp-state and observed that the probe side chain is generally exposed and free, without obvious side chain interactions that could potentially introduce artifacts.At the same time, we do observe that in some frames of the simulation trajectory, the finger loop has a tendency to form a helical-like structure, which is expected because similar structures are observed in a number of cryo-EM structures.This result is also included in the Supplementary Fig. 9 and also attached here.(From Supplementary Fig. 9 of the revised manuscript) Furthermore, we performed 19 F CEST experiment to provide direct evidence for the interchange between these conformation on a slow NMR timescale (shown in Supplementary Fig. 13 of the revised manuscript and also in Fig. R8 above).
In the revised manuscript, we have shortened this section so that the main text focus on presenting the NMR observations.The original figure panels of the different finger loop conformations observed in the crystal and cryo-EM structures are moved into the Supplementary Information file.Discussion on correlating the NMR peaks with the crystal structures is revised and shorted (page 8-9 of the revised manuscript) as follows: "… Among the activated βarr1 structures, the finger loop flips up and most commonly adopts a random coil conformation that allows L71 to insert into the cytoplasmic cavity of the receptor core.Moreover, a distinct finger loop conformation, in which it not only flips up but also forms a small helix between residues R65 and L71 is observed in both neurotensin receptor 1 (NTSR1) -complexed βarr1 structures (PDB: 6UP7 and 6PWC).Similar helical-like conformation is also observed in the MD simulation trajectory of the 19 F-labeled βarr1 at the L71C site (Supplementary Fig. 9d-e Response: We thank the reviewer for the comment.The effect of PIP2 binding on the finger and gate loops was described in the Cell paper by Janetzko et al. (2022).It was found that PIP2 binding does not trigger CT release but induces conformational changes at the finger loop and gate loop region to a lesser extent than V2Rpp.However, it remains unclear what kind of conformational state PIP2 actually stabilizes, whether it is "the same active state of βarr1 achieved with V2Rpp, albeit to a lesser extent" or "an active-like state of βarr1 that is onpathway toward activation and capable of binding Fab30" as the authors stated.Our NMR data coincide with the FRET and biochemical results, while at the same time provide a more detailed picture of the PIP2-stablized conformation at residue level.For example, for the five labeling sites at the central crest region, PIP2 perturbs only two at the back side of the βarr1 structure but not the three in the front, which is significantly different from the V2Rpp-binding effect.
The spectra differences between V2Rpp-stablized and PIP2-stablized states demonstrate that the PIP2-induced conformation is a distinct one.
Moreover, following the reviewer's comments on "how the signal is transduced from the PIP2 binding site to e.g.E313", we probed additional labeling sites including D240C and C242, which are located on the surface of C-domain and in-between the PIP2 binding site and E313.
The results indicate that PIP2 binding leads to line broadening for both sites, suggesting that PIP2 binding probably leads to destabilization of a stretch of residues at the back side of βarr1 towards E313.Inspection of the crystal structures suggests a subtle perturbation of the β15-β16 packing, supporting the allosteric effect is transduced through the β-strands.To describe these new results, we have revised the corresponding section, as well as Fig. 4 and the Supplementary Fig. 13.The revised paragraphs (page 11-12 of the revised manuscript) are as follows: "… Therefore, we examined the effect of PIP2 on a few more labeling sites in the C-domain (Fig. 4a and Supplementary Fig. 12c).The results show that PIP2 binding has limited effect on the E206C, F277C and E283C sites (linewidths increase < 10%), all of which are located close to the N-, C-domain interface at either the front or the bottom side of the C-domain.
Instead, we observe the most significant line broadening at the E313C, D240C and C242 sites (about 50%, 75% and 50% increase of linewidths, receptively), indicating enhanced local dynamics.The E313C site is located in the back loop and close to the domain interface.Both D240C and C242 sites are located in the β15 strand and are closer to the PIP2 binding pocket.
These observations indicate that PIP2 induces enhanced dynamics for a cluster of residues involving the β15 strand, the C loop and the back loop at the back side of the C-domain (Fig.

4b).
A possible scenario is that PIP2 binding to the K232, R236 and K250 residues in the β15 and β16 strands perturbs the conformational stability of these β-strands, which propagates towards the neighboring C loop and back loop regions.Clues can be found from the local differences in the β15-β16 packing observed in the βarr1 structures bound to the receptor NTSR1 with or without a PIP2 molecule (Supplementary Fig. 12d-f).In the presence of a bound PIP2, a subtle sliding between the β15 and β16 strands is observed, and the position of the A247 residue is slightly shifted towards the PIP2 binding site, affecting the backbone contact between A247 and I241.This change can directly affect the C loop that links the β15 and β16 strands, thus can explain the line broadening at the F244C site.The local destabilization could also perturb the neighboring β18 strand and the back loop that connects to it.
Notably, the back loop residue E313 is adjacent to a previously identified "finger-loop proximal" region that comprises a cluster of charged residues (R76, K77, D78 in βarr1 and

Reviewer #2 (Remarks to the Author):
Via cysteine mutagenesis, the authors use 19F NMR to investigate conformational response of both a CT-truncated and full-length version of Beta-arrestin-1, as a function of various phospho-peptides, V2Rpp, and PIP2.The work builds upon a recent study by Janetzko et al, but with a focus on conformational ensembles.I think the work is interesting and above the bar for publication in Nature Commun.It might have been extremely interesting to attempt NMR spectra in the presence of a receptor but I certainly appreciate how much more difficult this would be.While for the most part, PIP2 and V2Rpp seem to act independently, the modest enhancement of the active signature at D390 for example, speaks to the inherent cooperativity and allostery present in the system.

Response:
We thank the reviewer very much for the useful suggestions.We quite agree with the reviewer's suggestion of using a full receptor to interact with βarr1 and we are really hoping to do this.However, the receptor sample that we can obtain at the current stage could not survive the required NMR experimental time.We fear that receptor instability during the data acquisition time can introduce artifact, and therefore we are not able to produce reliable datasets at the current stage.
Moreover, we agree with the reviewer's comment on the inherent cooperativity and allostery in βarr1 activation.In the revised manuscript, we have performed a number of additional experiments and largely revised the manuscript.The major changes including: (1) Conformational changes in the C domain upon binding V2Rpp (a new section titled "V2Rpp-induced changes in the C-domain", on page 9 of the revised manuscript); (2) Examination of the cross-talk between PIP2 and V2Rpp binding; (a new section titled "Cross-talk between phosphopeptide and PIP2 binding", on page 14-15 of the revised manuscript); (3) Examination of the effects of membrane binding on βarr1 using lipid nanodisc, which again reveals long-range allosteric effects (a new section titled "Effects of membrane binding on βarr1 conformational dynamics", on page 15-16 of the revised manuscript); (4) Further analysis of the mechanism by which PIP2 binding induced dynamics changes propagates to the back loop region (section "PIP2 induces βarr1 activation from the back side of the N-, C-domain interface", page 11-13 of the revised manuscript).
These results together demonstrate an allosteric linkage between the two domains, and provide more comprehensive implications of the highly complex conformational energy landscape of βarr1 during activation.Based on these results, we have also largely revised the manuscript discussion and corresponding figures.
In testing the class A and class B receptor responses and the connection to transient and long lived desensitization, is it sufficient to use V2Rpp or phospho-peptides from the Beta-2 adrenergic receptor or are there additional differentiators in the receptor itself?I am assuming that beta-Arr1 can undergo both transient and long-lived desensitization.In this case, it might be helpful to use something like alpha-fold to identify the class A and B complexes and point out interaction surfaces on Barr-1, how they might differ, and how this might be reflected in the current data.

Response:
We thank the reviewer for the question and suggestion.Regarding the different behaviors between the class A and class B receptors, contribution from the C-tail is more clearly established, whereas much less is understood about the contribution from the TM core interacting site.To better discuss this point, we did the following revisions: 1) In the section "Phosphopeptides from β2AR tail shows minimal effects on βarr1 conformation", we add a brief paragraph discuss why β2AR peptides fails to induce significant structural changes.We cited the related literature on the phosphorylation barcode required for strong activating effect, particularly the recent study that identifies a PxPP motif.We also collected the NMR data of the D390C site using a few V2Rpp mutants that removes certain phosphorylation sites, which provides supportive evidence for the importance of specific phosphorylation pattern in the V2Rpp, which are absent in the β2AR peptides.This data is added as panel d of Fig. 3 in the revised manuscript, and also attached here.We revised the corresponding paragraph (page 9-10 of the revised manuscript) as follows: "These observations are not surprising, because growing evidence have suggested the importance of correct phosphorylation barcoding (27)(28)(29)(30), and particularly a recently identified P-X-P-P motif to be essential for activating β-arrestins (18).Our NMR data of 19 Flabeled βarr1 titrated with V2Rpp mutants with certain phosphates removed also support the essential role of the 5th phosphorylation site and the cluster of phosphates at the 6-7-8 sites (Fig. 3d).Such motifs, however, are not present in the sequence of the β2AR peptides with GRK2 or GRK6 phosphorylation patterns." 2) As for the question of whether there are additional differentiators in the receptor core itself for determining transient or long-lived desensitization, we consider it highly possible but remains rather elusive based on currently available data.In the figure below we show the receptor core-finger loop contact interface of a number of available GPCR-βarr1 complex structures.Strong structural variations are observed among these structures, including not only the finger loop conformation but also the relative orientation of βarr1 to the receptor.In all cases, the short fragment of E66-L73 of the finger loop appear to be essential in engaging the TM core binding pocket, stabilized by hydrophobic and charged interactions.However, detailed interaction modes differ from one another.These observations, as well as the fact that the number of GPCR-arrestin complex structures is still very small, makes it hard to propose a hypothesis of how the TM core itself encodes selectivity.We tried examining the experimentally-determined and alpha-fold structures but did not obtain helpful information at the current stage.

Fig. R13 Comparison of the receptor TM core-finger loop interface among available complex structures.
Moreover, because of the technical difficulties, examination of receptor core-βarr1 interaction is not performed in our current study, and therefore we do not have experimental data to directly provide insights regarding this question.Nevertheless, we added a brief sentence in the Discussion section of the revised manuscript (page 18) to point out this possibility: "…While the correlation between the phosphorylation barcode of receptor C-tails and the functional outcome has been extensively investigated (27)(28)(29)(30), it is yet elusive whether the receptor TM core-finger loop contacting site encodes additional conformational/functional selectivity due to the scarcity of available structures." Minor comments: 1. Abstract -" We further show that the membrane phosphoinositide PIP2 independently modulates βarr1 conformational dynamics without displacing its autoinhibitory carboxyl tail, leading to a distinct partially activated state."Unless I am mistaken, this was the conclusion of the Cell paper by Janetzko et al.This sentence should therefore be expressed as a validation from this work rather than a new independent discovery.

Response:
We thank the reviewer very much for pointing this out, and we apologize for the inappropriate way of expression.During the revision, we have added more results including the CEST experiments and cross-talks between different binding partners, and therefore the abstract is largely revised.
In the Cell paper by Janetzko et al., PIP2 binding was found to be unable to trigger CT release and induces conformational changes at the finger loop and gate loop region to a lesser extent than V2Rpp by using FRET method, however, exact what kind of conformational state was stabilized by PIP2 remains unknown, as the authors stated in their paper: "While PIP2 may stabilize the same active state of barr1 achieved with V2Rpp, albeit to a lesser extent, it may also act to stabilize an active-like state of barr1 that is on-pathway toward activation and capable of binding Fab30, although to a lesser extent than V2Rpp-bound barr1.Further studies will be necessary to distinguish these possibilities."For the conclusion about PIP2 binding in the abstract, we revised the sentence into "… binding of the membrane phosphoinositide PIP2 stabilizes a distinct partially activated conformational state."Here we emphasize that the PIP2induced conformational state is a distinct one, which is clearly demonstrated by the NMR data but not quite obvious from the previous Cell paper.In this way we hope to clarify the new insight obtained from our current study, while at the same time avoid repeating the previous discovery by Janetzko et al.
Moreover, we added a sentence summarizing the new results at the end of the abstract as follows: "Our results further unveil a sparsely-populated activation intermediate as well as complex cross-talks between different binding partners, implying a highly multifaceted conformational energy landscape of βarr1 that can be intricately modulated during signaling."2. Fig. S2A."The data suggest that while all states show a certain degree of structural dynamics, the V2Rpp-bound state exhibit the highest heterogeneity with many sites displaying two or more conformations (Supplemental data Fig.S2a).In particular, residues in the central crest adopt a uniform conformation in the basal state, while they show multiple conformations when activated." Heterogeneity is harder than this to describe.Configurational entropy for example (sum of all n states pn*ln(pn)) might be one way but this excluded heterogeneity of a given state which could in principle be a function of the linewidth of each state (although we'd need to know the homogeneous and heterogeneous contributions).The point is that heterogeneity is not well defined here.Perhaps the authors were talking about heterogeneity between different domains?This is also something better answered by T2 relaxation experiments.Either way, this should be more rigorous.

Response:
We thank the reviewer for this comment and we agree that the "heterogeneity" is not rigorously defined.To avoid confusion, as well as to accommodate to the journal's requirement for manuscript length, we revised and shortened this part to a single sentence describing the observation of multiple separate peaks in the V2Rpp-activated state: "In general, two or more separate peaks are observed in the spectra of the V2Rpp-bound state for many structural regions, suggesting the existence of multiple conformations in slow exchange with each other."We also compared the linewidths of the major peak for all sites in different functional states, which also contains information of possible exchanges between different conformations.We added a sentence in the revised manuscript (page 5) as follows: "Moreover, 3. Grammar.The paper is well written but there are some awkward or grammatically incorrect wording sentences: Response: We thank the reviewer very much for pointing out these mistakes.We have revised the manuscript according to the reviewer's comments.
-abstract "evidences highlight" Response: We have modified it into "evidence highlights" in the revised manuscript.
-page 4 "partially activation" Response: We have revised it into "partial activation".
-page 7 "site exhibit more" Response: The corresponding sentence is removed in the revised manuscript, but we have checked the manuscript to avoid similar grammar mistakes.
-page 11 "involvement in [the] Barr activation pathway" Response: We have revised it into "involvement in the βarr activation pathway" as the reviewer pointed out.
-page 13 "the distal CT regions does modulate" Response: The corresponding sentence is removed in the revised manuscript, but we have checked the manuscript to avoid similar grammar mistakes.
-page 14 "the structural and dynamics changes" Response: The corresponding sentence is removed in the revised manuscript, but we have checked the manuscript to avoid similar grammar mistakes.
-page 15 "combinatory effects" Response: We have revised it into "combined effects".
-page 24 "states crystal structures" Response: We have revised the sentence into "The crystal structures of βarr1 in the inactive (PDB: 1JSY) and V2Rpp-activated (PDB: 4JQI) states are shown …".

REVIEWER COMMENTS
Reviewer #2 (Remarks to the Author): Based one the extensive revisions by the authors and differenfiafion of the new manuscript, I think it's suitable for publicafion.

Reviewer #3 (Remarks to the Author):
Here, the authors use 19-F NMR to monitor local conformafional changes in beta-arresfin 1 in response to different phosphopepfide tails.This research is generally important to the field, as many prior studies have used the high-affinity vasopressin receptor tail to drive complete arresfin acfivafion.My primary experfise is in molecular dynamics simulafion; as such, I am going to comment mainly on the MD-focused aspects of the manuscript, which were added during the revision to the manuscript.
The authors perform simulafions of V2Rpp-bound and full-length beta-arresfin 1 with the 19-F NMR label covalently incorporated into several sites of interest.Simulafions are several hundreds of nanoseconds in length and use reasonable parameterizafion schemes for modeling the label.The simulafion data are used to support the nofion that labels do not directly interact with nearby residues.I recommend that the authors quanfitafively demonstrate this, perhaps by using analysis codes to idenfify hydrogen bonds or other types of non-covalent interacfions between the label and nearby sites.(Supplementary Fig. 5 is primarily based on structural snapshots of these data.)My other recommendafion is that the authors raise the caveat that MD simulafions on these short fime scales cannot rule out the possibility that introducing labels at these sites of interest does not perturb the conformafional landscape on longer fimescales.However, this caveat likely applies to any number of biophysical techniques that incorporate labels into local sites to monitor behavior.An addifional way to interrogate these effects would be to demonstrate experimentally that any number of key properfies of arresfin (binding ability, etc) are not disrupted by labeling at those sites, but this is likely outside the scope of this manuscript.

Comments
The manuscript enfitled "Disfinct acfivafion mechanisms of β-arresfin 1 revealed by 19F NMR spectroscopy" by Zhai et al. studied the dynamic acfivafion mechanism of arresfin by using 19F NMR.The GPCR receptor-inifiated acfivafion of arresfin is known as a complicate process and many factors, such as receptor conformafion and posftranslafional modificafion, lipid binding and membrane anchoring, are involved in this process.The acfivafion is highly dynamic and, thus, the stafic structures only provide snapshots and parfial informafion of the process.Elucidafing such mulfi-conformafion dynamics is very challenging.The authors have used 19F NMR, which is very sensifive to protein conformafional changes, to monitor the arresfin acfivafion.First, the authors prepared various 19Flabeled beta-arresfin 1 proteins using their recently developed 19F probe and verified that the 19F labeling on selected sites didn't significant perturb the structure and funcfion by CD, Fab30 binding, clathrin binding, and molecular dynamic simulafion.The authors have then used chemically synthesized phosphorylated C-terminal pepfide of V2 receptor and beta2 adrenergic receptor to mimic the acfivated receptors.A preacfivated state has also been employed by C-terminal truncated arresfin.From the 19F NMR spectra recorded under various condifions, the authors idenfified that the 19F labels in different regions have differenfial responses.Of parficular interest is that the 19F NMR clearly showed PIP2 binding dramafically modulates the arresfin acfivafion and an intermediate state was idenfified by both 1D 19F spectra and 19F-CEST measurement.The authors have also observed that membrane binding of C can allosterically modulate the arresfin conformafional equilibrium.Although 19F NMR is powerful to study mulfi-state dynamics, it is known to provide limited structural informafion.Therefore, the peak assignment needs to be caufious.The authors recorded the spectra under different funcfion-related condifions and those changes are well consistent with the current funcfional and structural understandings.At least two labeling sites are chosen at each arresfin funcfional domain/regions and the results further indicated the observed spectral change are funcfion relevant.The authors also pointed out some potenfial limitafions of the current experiments and gave reasonable descripfions and discussions.Overall, this work beaufifully deciphered the conformafional dynamics of different regions of beta arresfin 1 upon interact with various factors.The manuscript is well organized, the results are very convincing, and the new findings are very interesfing and give invaluable dynamic view of the acfivafion mechanism of beta arresfin 1.Therefore, I highly recommend the manuscript to publish on Nat.Comm.Some suggesfions: 1. References are needed in line 132.2. Line 171, "in three states" may replaced with "under three condifions".3. Line 494, references are needed to support the statement.4. The authors may need add some more detail about the V2Rpp beta-arr1 binding experiment.What equafion are used for fifting? 5.The author may give some more details of the sefting up for 19F NMR experiments.Why is the recycle delay set to only 100ms?Are you using an Ernest angle for excitafion? 6.In line 126, "faster tumbling".isa liftle confusing with the whole protein tumbling.I suggest to use "reflecfing a highly flexibility" or "faster mofions", or others.

RESPONSE TO REVIEWER COMMENTS Reviewer #2 (Remarks to the Author):
Based one the extensive revisions by the authors and differentiation of the new manuscript, I think it's suitable for publication.

Response:
We thank the reviewer very much for the all the comments and suggestions, which greatly helped us improve this work.

Reviewer #3 (Remarks to the Author):
Here, the authors use 19-F NMR to monitor local conformational changes in beta-arrestin 1 in response to different phosphopeptide tails.This research is generally important to the field, as many prior studies have used the high-affinity vasopressin receptor tail to drive complete arrestin activation.My primary expertise is in molecular dynamics simulation; as such, I am going to comment mainly on the MD-focused aspects of the manuscript, which were added during the revision to the manuscript.
The authors perform simulations of V2Rpp-bound and full-length beta-arrestin 1 with the 19-F NMR label covalently incorporated into several sites of interest.Simulations are several hundreds of nanoseconds in length and use reasonable parameterization schemes for modeling the label.The simulation data are used to support the notion that labels do not directly interact with nearby residues.I recommend that the authors quantitatively demonstrate this, perhaps by using analysis codes to identify hydrogen bonds or other types of non-covalent interactions between the label and nearby sites.(Supplementary Fig. 5 is primarily based on structural snapshots of these data.) Response: We thank the reviewer very much for the suggestion.In the revised manuscript, we analyzed the potential hydrogen bonds and contacts between the probe and neighboring residues as the reviewer suggested.Potential hydrogen bonds are identified in very limited number of frames throughout the whole simulation trajectories, and they occur sporadically and transiently.Close contacts between the probe and nearby residues were also summarized and compared with the corresponding distances in wild-type protein structures.These data support the notion that the introduced 19 F probe is not likely to form artificial contacts, and are summarized in the Supplementary Tables 2-3.We also modified the Supplementary Fig. 5, in which we now plot the sidechain positions of all simulation frames to show the continuous space that the probe samples.To give a better description of these data, we add a paragraph "MD simulations of 19 F-labeled βarr1" in the Supplementary Discussion section including the following content: "To support that introduction of the wPSP-6F label does not cause artificial contacts and perturb βarr1 local structures, we performed MD simulation of apo-βarr1 labeled at the T6C, L71C, H295C, L334C and D390C sites, as well as V2Rpp-bound βarr1 labeled at the L71C site.As summarized in Supplementary Table 2, potential hydrogen bonds are sporadically detected in a very limited number of frames in the simulation trajectories of all the labeling sites, and none of them are observed to stably exist.Furthermore, we calculated all close contacts (defined as the situation where any two carbon atoms from two residues are within 7 Å distance) observed in the simulation trajectories.In Supplementary Table 3, we list all residues (excluding residues at the +1 and -1 positions of the labeling site) that are observed to form close contacts with the labeled probe during the MD.To evaluate whether these contacts deviate from the native conformation of βarr1, we compared the distances observed in the available crystal or cryo-EM structures with the MD data (Supplementary Table 3).The results suggest that the distances between the specific residue pair in the native βarr1 structure fall within the distance range detected in the MD simulation.By plotting the locations of the probe sidechain in all simulation trajectories onto the βarr1 structure (Supplementary Fig. 5), we can see that the probe sidechain is uniformly distributed in a continuous space allowed by the local structure, and observe no apparent bias towards specific orientations.For labeling sites that are more exposed (e.g.L71C and L334C), we observe that the probe sidechain is flexible and samples a wide range of orientations.Therefore, the close contacts detected between the probe with nearby residues are most probably the results of fast sidechain motions.……" Correspondingly, we revised the manuscript Methods section to add a paragraph "Analysis of the simulation results" with the following content: "Calculation of the hydrogen bonds and non-covalent contacts were performed using the CPPTRAJ module in AMBER 54 .The default parameters were applied for hydrogen bond calculation, with an angle cutoff at 135° and a distance (acceptor to donor heavy atom) cutoff at 3.0 Å.For evaluating potential non-covalent interactions between the probe and other residues, we define contact (between two residues) as the situation where the distance between any two carbon atoms from two residues is less than 7 Å.Residues that are found to have contacts with the 19 F probe during the simulation trajectory are further analyzed." My other recommendation is that the authors raise the caveat that MD simulations on these short time scales cannot rule out the possibility that introducing labels at these sites of interest does not perturb the conformational landscape on longer timescales.However, this caveat likely applies to any number of biophysical techniques that incorporate labels into local sites to monitor behavior.An additional way to interrogate these effects would be to demonstrate experimentally that any number of key properties of arrestin (binding ability, etc) are not disrupted by labeling at those sites, but this is likely outside the scope of this manuscript.
This article by Zhai et al. describes a study by 19F NMR spectroscopy to follow the activation of beta-arrestin1 (βarr1) by phosphorylated peptides and by PIP2.The authors introduced the fluorine label PSP-6F, which carries 2 CF3 groups on a pyridine ring, by cysteine ligation to a large number of different cysteine mutants of full-length βarr1 as well as of a truncated βarr1 version (ΔCT, residues 1-382), which has the C-terminal strand β20 removed.The latter truncation is well known to facilitate activation as it removes the blocking of β1 for binding of GPCR phosphorylated C-terminal tails.The authors determine the NMR response of many single-site fluorine labels on full-length βarr1 and ΔCT to the addition of the well-studied phosphorylated C-terminal tail peptide V2Rpp of the V2 vasopressin receptor, two different phosphorylated C-terminal tail peptides from the β2-adrenergic receptor (β2AR), as well as to PIP2.They observe distinct changes in various regions of βarr1 (N-terminal, central, C-domain).In some cases, several lines 19F lines are observed upon V2Rpp binding, from which the authors conclude a heterogeneity of conformations.The β2AR peptides have no significant effect.However, PIP2 binding to the Cdomain (abrogated by the 3Q mutations in the C-domain) induces spectral changes in the central 'crest' region, which is the hinge between the N-and C-domain.

Fig. R1
Fig. R1 Comparison of the chemical structure of the wPSP-6F with several other probes.

Fig. R2 1
Fig. R2 1 H NMR verification of the structural integrity of the 19 F-labeled βarr1 samples.(SupplementaryFig.2of the revised manuscript) (a) 1 H NMR spectra of the 19 F-labeled βarr1 samples compared with the wild-type full-length protein showing the amide signal region.The five 19 F-labeled samples shown at the bottom (D240C, C242, C269, H198C and L191C) were prepared using a different batch of buffer in which cocktail protease inhibitor, TFA and DTT were pre-added, and was therefore compared with a WT sample using the same buffer.The strong peaks in the 7.5-7.7 ppm region originate from the small molecules.(b) 1 H NMR spectra of the 19 F-labeled βarr1-ΔCT samples compared with the wild-type βarr1-ΔCT showing the amide signal region.

Fig
Fig. R3 CD spectra verification of the structural integrity of the 19 F-labeled βarr1 samples.(Supplementary Fig. 3 of the revised manuscript) (a) CD spectra of the 19 F-labeled βarr1 samples compared with the wild-type full-length protein.(b) CD spectra of the 19 F-labeled βarr1-ΔCT samples compared with the wild-type βarr1-ΔCT protein.

Fig. R4
Fig. R4 Functional verifications of the 19 F-labeled βarr1 samples.(Supplementary Fig. 4 of the revised manuscript) (a) Ni-affinity pull-down assays showing the V2Rpp-enhanced binding to Fab30 by the wildtype and 19 F-labeled βarr1 proteins.(b) GST pull-down assays showing the V2Rpp-enhanced binding to clathrin by wild-type and 19 F-labeled βarr1 proteins.

Fig
Fig. R5 Molecular dynamics simulations of 19 F-labeled βarr1.(Supplementary Fig. 5 of the revised manuscript) (a) Overall structural comparison between representative conformers of the 19 F-labeled βarr1 at five critical labeling sites (dark grey) compared to the wild-type protein (dark green).The representative conformers are derived from structural frames showing the median RMSD values from the starting structures.(b-f) MD simulation results of the local structures (from randomly selected simulation frames) showing the conformational space that the 19 F-probe sidechain may sample (grey stick representation) compared to the wild-type protein (green).No apparent artificial interactions between the probe side chain and nearby residues are observed during the simulations.

Fig. R6
Fig. R6 Verification of the dual peaks at the T6C site labeled with different 19 F-probes.(FromSupplementary Fig.7of the revised manuscript)

Fig. R8 19 F
Fig. R8 19 F CEST experiments verifying conformation exchanges in βarr1.(Supplementary Fig. 13 of the revised manuscript) (a-b) 19 F CEST profiles of the L71C site in the V2Rpp-bound (a) and ΔCT states (b) of βarr1.In both cases, peak intensity ratios were calculated using either S1 or S3 as the reference.(c-e)

Fig. R9
Fig. R9 Summary of the apparent peak numbers and linewidths of βarr1 in different states.(FromSupplementary Fig.6of the revised manuscript)

Fig
Fig. R11 MD simulation results of the wPSP-6F-labeled L71C mutant in the V2Rppbound state showing the full structure (d) and the finger loop region (e).The local helicallike conformation observed in the simulation trajectory is indicated.(From Supplementary Fig. 9 of the revised manuscript) ).The structural diversity observed in the crystal or cryo-EM structures echoes with the complex NMR spectra in the finger loop region.The S2 and S3 resonances of L71 induced by V2Rpp binding represent two different activated conformations (or conformational ensembles) that may correspond to the random coil or helical structures, or may represent alternative activation intermediates.In either case, the fact that the finger loop samples multiple conformations when activated underlies the plasticity of βarrs in binding to diverse GPCRs."(iv) Figure 5+6: the effect of PIP2 binding on the hinge region between N-and C-domain has recently been described in detail by Janetzko et al. (2022) using FRET and fluorescence reporters on the finger and gate loops.Again, structural changes of the residues shown as perturbed in Figure 6b are completely expected.The question remains unanswered how the signal is transduced from the PIP2 binding site to e.g.E313.

Fig. R12
Fig. R12 Effect of different phosphorylation patterns on CT release.(From Fig. 3 of the revised manuscript)

a
majority of the structural regions (except for the CT) show increased resonance linewidths in the ΔCT or V2Rpp-bound states, further supporting enhanced dynamics (e.g., intermediatetimescale exchange between multiple conformations that are subtly different from each other) when βarr1 becomes activated."In addition, we performed 19 F CEST experiments during the revision to provide further information about conformational exchanges.For one thing, the CEST profiles help confirm that the multiple resonances observed in the 1D spectra are in exchange with each other.For another, The CEST results help unveil a low-populated intermediate conformation in various structural regions.We added a new section " 19 F CEST data unveils an intermediate conformational state" in the revised manuscript to describe these results.