1. Biophysics Colab

    Authors' response (16 December 2021)

    GENERAL ASSESSMENT

    The TMEM16 family of membrane proteins have been shown to function as calcium-activated chloride channels and lipid scramblases. In recent years, X-ray and cryo-EM structures have been solved for TMEM16 proteins in ligand-free and ligand-bound conformations, providing valuable structural information on their functional duality and activation mechanisms. It is largely accepted that the catalytic site (termed subunit groove or cavity) is mostly shielded from the membrane in the ligand-free TMEM16 scramblases. Calcium binding induces a conformational rearrangement of the cavity-lining helices, opening the groove to the surrounding membrane. Since the groove is hydrophilic, it was proposed that it serves as a permeation pathway for lipid headgroups while the hydrophobic lipid tails remain embedded into the hydrophobic membrane core, which has been termed the "credit card" mechanism of lipid scrambling. Additionally, structures of several TMEM16 homologs in lipid nanodiscs revealed that these proteins deform the lipid bilayer in the vicinity of the subunit cavity by bending and thinning the membrane, irrespective of the presence of the activating ligand calcium. Functional experiments also suggested that lipids can be scrambled outside of the open subunit cavity and that local protein-induced membrane deformation is critical for lipid scrambling.

    In the present study, Falzone and colleagues further address the mechanisms of lipid scrambling using single particle cryo-EM and liposome-based functional assays. Firstly, the authors solved the structure of a calcium-bound fungal homolog, afTMEM16, in nanodiscs with a lipid composition where the protein is maximally active. Although similar structures were obtained before, this new structure has the highest resolution thus far, representing > 1 Å improvement! The structure is beautiful and is a major achievement, which enabled the authors to resolve individual lipids and their interaction with the protein around the subunit cavity, whereas in previous structures unresolved non-protein densities were observed passing through the groove. The authors also solved a number of structures with and without calcium in lipid compositions that promote (thinner lipid bilayers) or suppress (thicker lipid bilayers) scrambling. The authors show that afTMEM16 can scramble lipids while the subunit groove remains closed, a phenomenon that is further enhanced in thinner membranes, whereas in thicker membranes scrambling is suppressed even though the groove is open. We particularly appreciated how different software packages and processing strategies were used to rigorously identify structural heterogeneity in their cryo-EM data. Remarkably, mutations of residues lining the subunit cavity and interacting with lipids do not appear to have dramatic effects on scrambling rates, which suggests that lipids do not need to interact with the protein to be scrambled. Thus, the overall conclusion of the study is that membrane thinning by TMEM16 scramblases in their calcium-free conformation is enough to induce lipid scrambling, and that the groove opening induced by calcium binding further enhances membrane deformation, promoting faster scrambling. By contrast, in thicker membranes the protein fails to sufficiently deform the bilayer and scrambling is suppressed, even when the subunit groove is open. The present study provides unprecedented structural information on the interaction of lipids with afTMEM16 and new evidence that lipids can be scrambled outside of the groove.

    The findings and conclusions presented here help to explain why TMEM16 scramblases can transport lipids with headgroups much bigger than the dimensions of the subunit cavity and why structures of some of the other scramblases (opsins, Xkrs and mammalian homolog TMEM16F) lack the obvious hydrophilic groove seen in fungal TMEM16 scramblases. Overall, this is a well-rounded study with an exceptional amount of high-quality cryo-EM data and functional experiments supporting the conclusions.

    We thank the reviewers for their praise of our work and for their constructive criticisms. Below we provide a detailed response to their comments and suggestions.

    RECOMMENDATIONS

    Revisions essential for endorsement:

    1. The resolved lipids binding within the groove in the first structure might be seen by some as supporting the credit card mechanism as it definitively demonstrates that lipids reside within the groove. While the authors provide evidence that lipids can permeate outside the groove in this and earlier work, as far as we can tell, none of that would preclude permeation through the groove if it doesn't require specific interactions between lipids and sidechains in the protein. The presentation might be improved with a somewhat more circumspect and nuanced exposition of the new data and how it can be understood with earlier results.

    There are several reasons why we do not think that the lipids in the C18/Ca2+ structure support the credit card mechanism, at least in the incarnation proposed for the TMEM16 scramblases.

    1. In the credit card model, lipid headgroups enter and traverse the whole span of the groove (as described in multiple publications, i.e. Bethel and Grabe, PNAS, 2016; Jiang et al., Elife, 2018; Lee et al., Nat Comms, 2018; Kostriskii and Machtens, Nat Comms, 2021). The lipid densities near the groove suggest that P3 and P4 lipids are oriented with their heads facing the groove's exterior, not the interior. These heads are contiguous with other resolved lipids in the outer and inner leaflets, respectively. We added panels showing views of the pathway from the extracellular solution to better convey that the lipid heads do not enter the groove (see new Fig. 1F-G). We also added a statement on pg. 10 to clarify this important point.
    2. In the present structures, which are consistent with earlier ones with lower resolution (Falzone et al., Elife, 2019; Kalienkova et al., Elife, 2019), residues in the extracellular vestibule do not interact with lipids (see new panels 1E-G). In contrast, the wide intracellular vestibule is embedded in the membrane. We agree with the reviewers that lipid headgroups can, and likely will, enter this wide vestibule during scrambling. We modified the text on pg. 12 to clearly state this point "The wide intracellular vestibule is embedded in the nanodisc membrane and, at the open pathway, the resolved P3 and P4 lipids have opposite orientations (Fig. 2A), suggesting scrambling might occur between them. In this case, the lipid headgroups would only need to move through the wide intracellular vestibule of the pathway below the T325-Y432 constriction rather than through the whole groove (Fig. 2A)."

    These observations, together with the extensive mutagenesis data reported in Fig. 2 and 3, point to a mechanism that is different from the precisely coordinated credit-card mechanism that is the currently accepted paradigm for lipid scrambling.

    Might the complex composition of native lipid membranes influence where and by what mechanism lipid movement between leaflets is catalysed by TMEM16 proteins?

    The idea that the lipid composition might affect the mechanism of scrambling (i.e. through the groove vs out of the groove) is very interesting, and we are actively investigating it in the lab. However, it would be surprising if different lipids were scrambled by entirely different mechanisms.

    1. The quality of lipid densities in cryo-EM structures is greatly affected by the number of particles used and the resolution obtained during refinement and it is therefore not surprising that the beautiful lipid densities observed here in the structure of afTMEM16 in lipid nanodiscs in the presence of calcium refined to 2.3 Å are not all observed in subsequent structures with lower resolution. This is true not only for the P lipids near the groove, but for those D lipids bound near the dimer interface, which is a stable region of the protein that does not change conformation. To be cautious, the authors should avoid resting any conclusions on the absence of lipid densities in the lower resolution structures. For example, on pg 15 the authors seem to be interpreting the absence of density for C22 lipids.

    We agree with the reviewers on this point. However, at ~2.7 Å average resolution and with >130,000 particles we would expect to see density for lipids near the pathway, if these were tightly bound. For example, in the mTMEM16F nanodisc structures from the Chen and Jan labs (Feng et al., Cell Reports, 2020), several lipid densities were identified near the closed pathway despite a substantially lower average resolution. However, we agree that we should not interpret this lack of signal and toned down our statement, "This suggests that the interactions of C22 lipids with the pathway helices are weaker than those of C18 lipids, possibly reflecting a higher energy cost associated with distorting these longer acyl chain lipids" to better indicate this is a possible explanation, rather than a definitive mechanistic interpretation.

    1. The presentation of structural interactions between lipids and residues near the groove of the protein could be improved in the figures. A panel like Fig. 1J but for the groove would help, but it would be good to see expanded perspectives in the form of a supplementary figure where residues around the headgroup of the lipids are shown along with EM maps so the quality of the structural information for both lipids and side chains can be better appreciated. The preprint does have a lot of images of the lipids and the protein, but not in a way that enables the reader to quickly grasp the nature of interactions between side chains and lipid moieties for themselves, and we feel that close-ups of individual lipids as suggested above would help.

    We thank the reviewers for this suggestion. We show density maps for the protein and lipids in Fig. 1C-E, and added close-up views of the densities near the groove in the new Fig. 1F-G to highlight the poses adopted by the lipids in this region. Figures showing both density and atomic models for the protein and lipids are very busy and difficult to discern; many of the lipids interact with multiple residues from different helices, with both their heads and tails. As such we could not find satisfactory views displaying both for the majority of the lipids.

    It is also not clear what the authors mean by "lipid headgroup". Have the authors only considered interactions of the phospholipid phosphate group with protein residues? It would be helpful if the authors could clarify this in the manuscript and say whether other types of interactions were considered.

    In our C18/Ca2+ map, we resolve a total of 16 lipids per monomer. Of these, we assigned 2 as PG lipids, because we could resolve the large PG headgroup (D4 and D5), shown in Fig. 1F-H and Supp. Fig 2. In all other cases, we truncated the lipids at the phosphate, as the density was insufficient to distinguish between a PC and a PG headgroup. This is now specified in the Fig. 1 legend. In our mutagenesis experiments (Fig. 2 and 3), we only targeted residues that were within interaction distance of the resolved portions of the headgroups, which is the phosphate in most cases. This is now clarified on page 11 "we investigated how mutating residues coordinating the resolved portions of the headgroups of P1-2 and P4-6 impacts scrambling."

    It would also be nice to include a close-up view of D511A/E514A in 0.5 mM calcium with cryo-EM density to demonstrate the absence of bound calcium ions.

    We thank the reviewers for this suggestion. We added a new panel in Supp. Fig. 10H showing a close-up of the cryoEM density of the mutant binding site.

    1. The functional data in Fig 2, 3 and 4 are also not discussed in much detail and it would help if the authors could expand the presentation. Although scrambling in the presence of a very high concentration of calcium is not dramatically altered by any of the mutations, there is quite a lot going on in the absence of calcium and very little is said about these results. For example, differences in the scrambling rates can be observed with some mutants in the presence and absence of calcium in figures 2E and 3E, but statistical analysis would be required to know if the differences between mutants are significant. The differences in scrambling rates with different lipids are also not discussed (e.g. Fig. 4A) It would help if the authors could discuss what is the margin of error in the scrambling assay, and point to some concrete examples from their earlier work on this specific scramblase where mutants have a large impact on scrambling activity in their assay.

    We agree with the reviewers that most mutants show some effects in 0 Ca2+. The effects are statistically significant for all but one mutant (2-tailed t-test, p<0.005). However, the magnitude of the effects is relatively small (<7-fold reductions in all cases). While our approach to quantify the scrambling rate constant captures well large changes, some of the assumptions underlying the analysis make it less well suited to quantify small effects. In past publications we used a 10-fold change as a cut-off threshold to consider an effect meaningful (Lee et al., Nat comms, 2018; Khelashvili, Falzone et al., Nat Comms, 2020). These limitations and rationale for choices are discussed in several of our past publications (Malvezzi et al., PNAS, 2018; Lee et al., Nat Comms, 2018; Falzone and Accardi, MiMB, 2020). We added statements indicating magnitude of the observed reduction for the mutants in the various conditions. We prefer to refrain from presenting statistical significance of these results as we do not want to convey the idea these effects are more meaningful than they might be.

    Have the authors tried intermediate more physiologically relevant concentrations of calcium to see if the mutants have discernible effects under those conditions?

    This is an excellent suggestion. However, in our experience the technical limitations of the experimental set-up and of the analysis render a precise quantification of small effects at intermediate Ca2+ concentrations not very reliable. For this reason, we did not pursue this further.

    1. It is quite intriguing that the mutations in the subunit groove of afTMEM16 have little effect on scrambling activity. The authors propose that the groove-lining residues are not directly involved in lipid coordination even though their structure suggests that they do and there is a wealth of functional studies and MD simulations on various other TMEM16 homologs suggesting otherwise.

    We are a bit confused by the reviewers' statement that our structure suggests that groove lining residues coordinate lipids. In our structures, the only two residues that directly line the open groove and coordinate lipids are T325 and Y432 (Fig. 2A). All other 23 residues tested either do not line the groove (9 residues mutated in Fig. 2) or do not interact with lipids (14 residues mutated in Fig. 3). The finding that mutating these residues has minor effects on scrambling suggests that interactions between lipids and these side chains is not required for scrambling.

    We agree that the overall lack of effect of the mutants is surprising, especially in light of past work. However, none of the scrambling assays (in vitro or cell-based) can distinguish between mutations that affect permeation from those that affect gating. All that is measured is whether and -to a degree- how well lipids are transported. As such, we propose that at least some of the functional effects could have been misinterpreted. We are currently testing this hypothesis in the lab.

    The discrepancy between our structural and functional results and the molecular mechanism emerging from MD simulations is more striking. Although some differences exist between the reports of different groups, the overall agreement among them is excellent. We were thus surprised that our data is so difficult to reconcile with their observations. Indeed, the extensive mutagenesis reported in Fig. 2 and 3 was performed to systematically test the unexpected inferences of our initial structural results (on the C18/Ca2+ structure). Our conclusions are also corroborated by the structures in different lipid compositions. In the discussion (pg. 21-22) we consider some of the possible sources for these discrepancies. For example, while in the MD simulations of nhTMEM16 the extracellular vestibule (i.e. E305, E310 and R425) is immersed in the groove, in our cryoEM maps we do not see evidence of lipids interacting with these residues (Fig. 1,2,3). Notably, a similar arrangement of the membrane-protein interface is seen in the Ca2+-bound open nhTMEM16 structure in nanodiscs (Kalienkova et al., Elife, 2019), indicating this issue is not specific to afTMEM16 or to the nanodisc used. We hypothesize this different membrane-protein interface is at the origin of the different proposed mechanisms. Another potentially relevant difference is that the tails of multiple lipids intercalate between helices forming the dimer cavity, some of which line the groove (Fig. 1). These lipids were not included in MD simulations as they were not previously resolved, and they could affect groove dynamics and, consequently, its interactions with the membrane. Other possibilities exist, but we believe they are less likely to be important (i.e. the limited nature of nanodiscs used for the cryoEM experiments could influence the protein-membrane interface, the mutations could have effects that are too subtle to measure in our assay). However, we think that enumerating all possibilities would lead to an overly lengthy discussion and require too much speculation.

    We have revised the discussion of these important points in pg. 21-23 to better convey these uncertainties and added a statement (pg. 11) where we report the distance between the phosphate atom of the P3 lipid and E305 (13.7 Å), E310 (17.9 Å) and R425 (15.7 Å).

    The authors' suggestion that mutations probably affect the equilibrium between open and closed conformations of the groove in other homologs but not in afTMEM16 is logical, however, there are some discrepancies. To name a few examples, if indeed this is the case, nhTMEM16 mutants with closed groove should still have significant basal scrambling, by extrapolation from afTMEM16 data. Yet, some of the nhTMEM16 mutants (E313/E318/R432 mutants) have no activity at all, or no basal scrambling activity (Y439A) (Lee et al, 2018). Would you expect that point mutations within the subunit groove remove the ability of the protein to deform the membrane in its closed conformation? Might the groove have intermediate conformations between closed and fully open where the mutants studied might have more impact in afTMEM16?

    These are excellent ideas, and we are actively pursuing them in the lab. However, at the moment results are too preliminary to draw firm conclusions.

    Further, mutating some of the residues on the scrambling domain of TMEM16 affected externalization of some lipid species, but not internalization etc. (Gyobu et al, 2017), which should not be the case if the interaction of the protein with the lipids is completely unnecessary for lipid scrambling.

    This is a good point. However, mechanistic interpretation of results from cell-based scrambling assays is quite tricky, even more so than of the results from the in vitro measurements used in the present work. The presence of other lipid transporters and/or scramblases, or a multitude of other factors, could influence the results. For example, in cells scrambling by TMEM16F is delayed, it takes ~10 minutes after Ca2+ exposure to begin seeing PS externalization. In contrast, in in vitro measurements TMEM16F responds to Ca2+ nearly instantaneously, within the ~1 s mixing time of the cuvette (Alvadia et al., Elife, 2019). Thus, a direct comparison of the results obtained in cells and in vitro is not straightforward. More work is needed to investigate these important points.

    While investigating this question further would require follow-up structural studies on other TMEM16 homologs and is outside of the scope of this study, we think that the manuscript would benefit from a more extensive discussion on contradicting results and alternative interpretations. The authors might want to consider the possibility that there may be substantial variations in how different scramblases function.

    We agree that it is a priori possible that different TMEM16 proteins function according to different paradigms. However, we think this is an unlikely possibility. Despite differences in their gating behavior, most basic functional properties of TMEM16s are well conserved. Thus, fundamentally different mechanisms (i.e. through the groove or out of the groove) would have to result in similar functional phenotypes. We find the hypothesis that the basic scrambling mechanism is conserved among different TMEM16 homologues more plausible. While our results do not rule out that through the groove scrambling can occur, they suggest that it is not the main mechanism for afTMEM16, despite the fact that this protein adopts a very stable conformation with an open groove. Therefore, we consider the possibility of different mechanisms unlikely. This is mentioned on pg. 22 of the discussion.

    afTMEM16 has high constitutive activity in the absence of calcium, while at least TMEM16F does not. Additionally, the extent to which scrambling is promoted by calcium varies, as mammalian scramblases might need other cellular factors to be activated. Also, the extent to which scramblases are seen to distort the membrane is highly variable, as again seen in TMEM16F structures. Might some of these differences imply that key aspects of the mechanism of scrambling (e.g. thinning of the membrane or whether lipids scramble inside or outside the groove) are not the same for all scramblases? This might be one way to organize the discussion to help reconcile some of the seemingly divergent findings in the field.

    The reviewers raise an excellent point. Indeed, we find that for all TMEM16 homologues we have tested in the lab the degree of activity in 0 Ca2+ is highly dependent on the lipid composition. However, this does not appear to correlate with changes in conformation, as we report here for afTMEM16 and as reported by other groups for nhTMEM16 and TMEM16F.

    1. The authors should correct the Ramachandran outliers in C18/calcium and C22/calcium structures.

    We tried fixing the Ramachandran outliers, however this invariably led to worse fits of the atomic models with the density. Therefore, we believe it is appropriate to leave them as they are.

    Additional suggestions for the authors to consider:

    1. In several instances the authors conceptualize hypothetical mechanisms to set up experiments and frame their interpretations, which is not always the most straightforward way to communicate findings and what they reveal. The 'conveyor belt mechanism' introduced on page 10 is never fully defined in a way that helps the reader to understand what the functional effects of the mutants teach us. Might it be easier to set up the experiment by asking whether the interactions between sidechains that apparently interact with lipid headgroups in the structure play a critical role in scrambling, present the results and then conclude that they do not appear to? Collectively the functional effects of mutants do appear to suggest that specific side chain interactions are not critical for scrambling, but the conceptualized mechanism here makes the conclusions come across as unnecessarily forced.

    We thank the reviewers for the suggestion. We agree that the conveyor belt mechanism is a bit of a strawman. However, it is a plausible mechanism based on the orientation of the lipids in the C18/Ca2+ map. The mutagenesis described in Fig. 2 was explicitly designed to test this possibility. Further, this allows us to draw a clear distinction between testing the roles of residues outside the groove and of side chains that directly line the groove.

    The credit-card mechanism has been formally introduced and discussed in the field but has already been shot down in earlier work from the group and seems overly simplistic if we already know that scrambling can occur both inside and outside the groove from earlier studies. Just something for the authors to think about.

    We do not believe our previous work (Malvezzi et al., PNAS, 2018) 'shot down' the credit-card model. While we proposed that the large, PEG-conjugated lipid headgroups traverse the membrane outside the groove, our model postulated that normal-sized headgroups were scrambled within the groove. Further, one of the recurring criticisms of that work, was that the path taken by the large PEG-conjugated lipids might not represent a physiologically relevant mechanism for normal lipids. Thus, the credit-card mechanism remained the dominant model to explain scrambling, as testified by many subsequent publications by multiple groups, including our own!

    1. The uninitiated reader would greatly benefit from more of an introduction to the functional scrambling assay in the results and material and methods section so they can understand the results being presented. In the Material and methods, the authors mentioned: "All conditions were tested side by side with a control preparation", perhaps add here what exactly served as control –wild type protein in C18 lipids? It would be valuable to include information on the reconstitution efficiency between their preparations (WT in different lipid compositions and WT vs mutants). these if possible.

    We thank the reviewers for this suggestion. We added a brief description of the assay in the Methods section and now specify that "All conditions were tested side by side with a control preparation of WT afTMEM16 reconstituted in C18 lipids."

    1. Also, does the C18/calcium cryo-EM structure have sufficient resolution to distinguish between specific phospholipids (PG or PC) at the D1-D9 or P1-P7 positions? It would be particularly valuable if the authors could comment on whether PG or PC are observed in the D and P positions, or which lipids are lining the groove (P3-P6).

    We could build 2 lipids as PG (D4 and D5), based on the presence of density that could accommodate the large PG headgroup. For other lipids, the density was too weak beyond the phosphate, and therefore we left them truncated. This is now stated in the Figure 1 legend.

    1. While not essential, it would be interesting if the authors could perform the assay on the mutants with a more prominent effect in the absence of calcium (e.g. E310A, Y319A/F322A/K428A) with several additional calcium concentrations.

    We thank the reviewers for this suggestion. However, as we noted above, given the relatively small effects and limitations of the assay, we do not believe we would be able to extract meaningful mechanistic information from these measurements in intermediate conditions.

    1. The authors mentioned that the interaction of C22 lipids with the pathway helices is weaker than those of C18 lipids, which reflects the energy cost associated with distorting the longer lipids (page 15). However, they claimed that the interaction between the lipids and residues is not important for scrambling, which seems contradictory.

    We apologize for the confusion. In our proposed model, the ability of afTMEM16 to thin the membrane is dictated by the interactions of the protein with the surrounding lipids. This is not only enabled by interactions between side chains and lipid headgroups, but also by interactions of the lipid tails interact with the protein (see for example the close-up panels in Supp. Fig. 2F-G and the text on pg. 11 "Rather, other factors, such as tail interactions with interhelical grooves, contribute to their association with afTMEM16 (Supp. Fig 2F-G) and stabilize the distorted membrane-protein interface that results in thinning at the pathway.")

    (This is a response to peer review conducted by Biophysics Colab on version 1 of this preprint.)

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  2. Biophysics Colab

    Consolidated peer review report (30 November 2021)

    GENERAL ASSESSMENT

    The TMEM16 family of membrane proteins have been shown to function as calcium-activated chloride channels and lipid scramblases. In recent years, X-ray and cryo-EM structures have been solved for TMEM16 proteins in ligand-free and ligand-bound conformations, providing valuable structural information on their functional duality and activation mechanisms. It is largely accepted that the catalytic site (termed subunit groove or cavity) is mostly shielded from the membrane in the ligand-free TMEM16 scramblases. Calcium binding induces a conformational rearrangement of the cavity-lining helices, opening the groove to the surrounding membrane. Since the groove is hydrophilic, it was proposed that it serves as a permeation pathway for lipid headgroups while the hydrophobic lipid tails remain embedded into the hydrophobic membrane core, which has been termed the "credit card" mechanism of lipid scrambling. Additionally, structures of several TMEM16 homologs in lipid nanodiscs revealed that these proteins deform the lipid bilayer in the vicinity of the subunit cavity by bending and thinning the membrane, irrespective of the presence of the activating ligand calcium. Functional experiments also suggested that lipids can be scrambled outside of the open subunit cavity and that local protein-induced membrane deformation is critical for lipid scrambling.

    In the present study, Falzone and colleagues further address the mechanisms of lipid scrambling using single particle cryo-EM and liposome-based functional assays. Firstly, the authors solved the structure of a calcium-bound fungal homolog, afTMEM16, in nanodiscs with a lipid composition where the protein is maximally active. Although similar structures were obtained before, this new structure has the highest resolution thus far, representing > 1 Å improvement! The structure is beautiful and is a major achievement, which enabled the authors to resolve individual lipids and their interaction with the protein around the subunit cavity, whereas in previous structures unresolved non-protein densities were observed passing through the groove. The authors also solved a number of structures with and without calcium in lipid compositions that promote (thinner lipid bilayers) or suppress (thicker lipid bilayers) scrambling. The authors show that afTMEM16 can scramble lipids while the subunit groove remains closed, a phenomenon that is further enhanced in thinner membranes, whereas in thicker membranes scrambling is suppressed even though the groove is open. We particularly appreciated how different software packages and processing strategies were used to rigorously identify structural heterogeneity in their cryo-EM data. Remarkably, mutations of residues lining the subunit cavity and interacting with lipids do not appear to have dramatic effects on scrambling rates, which suggests that lipids do not need to interact with the protein to be scrambled. Thus, the overall conclusion of the study is that membrane thinning by TMEM16 scramblases in their calcium-free conformation is enough to induce lipid scrambling, and that the groove opening induced by calcium binding further enhances membrane deformation, promoting faster scrambling. By contrast, in thicker membranes the protein fails to sufficiently deform the bilayer and scrambling is suppressed, even when the subunit groove is open. The present study provides unprecedented structural information on the interaction of lipids with afTMEM16 and new evidence that lipids can be scrambled outside of the groove.

    The findings and conclusions presented here help to explain why TMEM16 scramblases can transport lipids with headgroups much bigger than the dimensions of the subunit cavity and why structures of some of the other scramblases (opsins, Xkrs and mammalian homolog TMEM16F) lack the obvious hydrophilic groove seen in fungal TMEM16 scramblases. Overall, this is a well-rounded study with an exceptional amount of high-quality cryo-EM data and functional experiments supporting the conclusions.

    RECOMMENDATIONS

    Revisions essential for endorsement:

    1. The resolved lipids binding within the groove in the first structure might be seen by some as supporting the credit card mechanism as it definitively demonstrates that lipids reside within the groove. While the authors provide evidence that lipids can permeate outside the groove in this and earlier work, as far as we can tell, none of that would preclude permeation through the groove if it doesn't require specific interactions between lipids and sidechains in the protein. The presentation might be improved with a somewhat more circumspect and nuanced exposition of the new data and how it can be understood with earlier results. Might the complex composition of native lipid membranes influence where and by what mechanism lipid movement between leaflets is catalysed by TMEM16 proteins?

    2. The quality of lipid densities in cryo-EM structures is greatly affected by the number of particles used and the resolution obtained during refinement and it is therefore not surprising that the beautiful lipid densities observed here in the structure of afTMEM16 in lipid nanodiscs in the presence of calcium refined to 2.3 Å are not all observed in subsequent structures with lower resolution. This is true not only for the P lipids near the groove, but for those D lipids bound near the dimer interface, which is a stable region of the protein that does not change conformation. To be cautious, the authors should avoid resting any conclusions on the absence of lipid densities in the lower resolution structures. For example, on pg 15 the authors seem to be interpreting the absence of density for C22 lipids.

    3. The presentation of structural interactions between lipids and residues near the groove of the protein could be improved in the figures. A panel like Fig. 1J but for the groove would help, but it would be good to see expanded perspectives in the form of a supplementary figure where residues around the headgroup of the lipids are shown along with EM maps so the quality of the structural information for both lipids and side chains can be better appreciated. The preprint does have a lot of images of the lipids and the protein, but not in a way that enables the reader to quickly grasp the nature of interactions between side chains and lipid moieties for themselves, and we feel that close-ups of individual lipids as suggested above would help. It is also not clear what the authors mean by "lipid headgroup". Have the authors only considered interactions of the phospholipid phosphate group with protein residues? It would be helpful if the authors could clarify this in the manuscript and say whether other types of interactions were considered. It would also be nice to include a close-up view of D511A/E514A in 0.5 mM calcium with cryo-EM density to demonstrate the absence of bound calcium ions.

    4. The functional data in Fig 2, 3 and 4 are also not discussed in much detail and it would help if the authors could expand the presentation. Although scrambling in the presence of a very high concentration of calcium is not dramatically altered by any of the mutations, there is quite a lot going on in the absence of calcium and very little is said about these results. For example, differences in the scrambling rates can be observed with some mutants in the presence and absence of calcium in figures 2E and 3E, but statistical analysis would be required to know if the differences between mutants are significant. The differences in scrambling rates with different lipids are also not discussed (e.g. Fig. 4A) It would help if the authors could discuss what is the margin of error in the scrambling assay, and point to some concrete examples from their earlier work on this specific scramblase where mutants have a large impact on scrambling activity in their assay. Have the authors tried intermediate more physiologically relevant concentrations of calcium to see if the mutants have discernible effects under those conditions?

    5. It is quite intriguing that the mutations in the subunit groove of afTMEM16 have little effect on scrambling activity. The authors propose that the groove-lining residues are not directly involved in lipid coordination even though their structure suggests that they do and there is a wealth of functional studies and MD simulations on various other TMEM16 homologs suggesting otherwise. The authors' suggestion that mutations probably affect the equilibrium between open and closed conformations of the groove in other homologs but not in afTMEM16 is logical, however, there are some discrepancies. To name a few examples, if indeed this is the case, nhTMEM16 mutants with closed groove should still have significant basal scrambling, by extrapolation from afTMEM16 data. Yet, some of the nhTMEM16 mutants (E313/E318/R432 mutants) have no activity at all, or no basal scrambling activity (Y439A) (Lee et al, 2018). Would you expect that point mutations within the subunit groove remove the ability of the protein to deform the membrane in its closed conformation? Might the groove have intermediate conformations between closed and fully open where the mutants studied might have more impact in afTMEM16? Further, mutating some of the residues on the scrambling domain of TMEM16 affected externalization of some lipid species, but not internalization etc. (Gyobu et al, 2017), which should not be the case if the interaction of the protein with the lipids is completely unnecessary for lipid scrambling. While investigating this question further would require follow-up structural studies on other TMEM16 homologs and is outside of the scope of this study, we think that the manuscript would benefit from a more extensive discussion on contradicting results and alternative interpretations. The authors might want to consider the possibility that there may be substantial variations in how different scramblases function. afTMEM16 has high constitutive activity in the absence of calcium, while at least TMEM16F does not. Additionally, the extent to which scrambling is promoted by calcium varies, as mammalian scramblases might need other cellular factors to be activated. Also, the extent to which scramblases are seen to distort the membrane is highly variable, as again seen in TMEM16F structures. Might some of these differences imply that key aspects of the mechanism of scrambling (e.g. thinning of the membrane or whether lipids scramble inside or outside the groove) are not the same for all scramblases? This might be one way to organize the discussion to help reconcile some of the seemingly divergent findings in the field.

    6. The authors should correct the Ramachandran outliers in C18/calcium and C22/calcium structures.

    Additional suggestions for the authors to consider:

    1. In several instances the authors conceptualize hypothetical mechanisms to set up experiments and frame their interpretations, which is not always the most straightforward way to communicate findings and what they reveal. The 'conveyor belt mechanism' introduced on page 10 is never fully defined in a way that helps the reader to understand what the functional effects of the mutants teach us. Might it be easier to set up the experiment by asking whether the interactions between sidechains that apparently interact with lipid headgroups in the structure play a critical role in scrambling, present the results and then conclude that they do not appear to? Collectively the functional effects of mutants do appear to suggest that specific side chain interactions are not critical for scrambling, but the conceptualized mechanism here makes the conclusions come across as unnecessarily forced. The credit-card mechanism has been formally introduced and discussed in the field but has already been shot down in earlier work from the group and seems overly simplistic if we already know that scrambling can occur both inside and outside the groove from earlier studies. Just something for the authors to think about.

    2. The uninitiated reader would greatly benefit from more of an introduction to the functional scrambling assay in the results and material and methods section so they can understand the results being presented. In the Material and methods, the authors mentioned: "All conditions were tested side by side with a control preparation", perhaps add here what exactly served as control – wild type protein in C18 lipids? It would be valuable to include information on the reconstitution efficiency between their preparations (WT in different lipid compositions and WT vs mutants). these if possible.

    3. Also, does the C18/calcium cryo-EM structure have sufficient resolution to distinguish between specific phospholipids (PG or PC) at the D1-D9 or P1-P7 positions? It would be particularly valuable if the authors could comment on whether PG or PC are observed in the D and P positions, or which lipids are lining the groove (P3-P6).

    4. While not essential, it would be interesting if the authors could perform the assay on the mutants with a more prominent effect in the absence of calcium (e.g. E310A, Y319A/F322A/K428A) with several additional calcium concentrations.

    5. The authors mentioned that the interaction of C22 lipids with the pathway helices is weaker than those of C18 lipids, which reflects the energy cost associated with distorting the longer lipids (page 15). However, they claimed that the interaction between the lipids and residues is not important for scrambling, which seems contradictory.

    REVIEWING TEAM

    Reviewed by:

    Angela Ballesteros, Research Fellow (K.J. Swartz lab, NINDS, USA): structural biology (X-ray crystallography), membrane protein function, lipid scrambling, cell biology, fluorescence microscopy

    Valeria Kalienkova, Postdoctoral Fellow (C. Paulino lab, University of Groningen, The Netherlands): membrane structural biology (X-ray crystallography and cryo-EM), membrane transport and lipid scrambling

    Kenton J. Swartz, Senior Investigator, NINDS, USA: ion channel structure and mechanisms, chemical biology and biophysics, electrophysiology and fluorescence spectroscopy

    Curated by:

    Kenton J. Swartz, Senior Investigator, NINDS, USA

    (This consolidated report is a result of peer review conducted by Biophysics Colab on version 1 of this preprint. Minor corrections and presentational issues have been omitted for brevity.)

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