Structural basis of closed groove scrambling by a TMEM16 protein

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1. 

   Biophysics Colab

   Feb 22, 2024

   Evaluation statement (17 January 2024; revised 31 January 2024)

   Feng and colleagues investigate the molecular basis of lipid scrambling in a fungal member of the TMEM16 family of Ca²⁺-dependent lipid scramblases. These proteins possess a groove in their 3D structure that has been implicated in lipid scrambling, which the authors investigate in the absence and presence of Ca²⁺ using a combination of cryo-EM structure determination, mutagenesis and functional assays. Their closed-groove structure reveals a continuous file of lipid molecules around the catalytic groove region, providing a structural basis for lipid interaction with the protein. Additionally, the authors capture three novel states of TMEM16, completing the picture of conformational transitions that this protein undergoes. Strikingly, the authors show that both structure and …

   Evaluation statement (17 January 2024; revised 31 January 2024)

   Feng and colleagues investigate the molecular basis of lipid scrambling in a fungal member of the TMEM16 family of Ca²⁺-dependent lipid scramblases. These proteins possess a groove in their 3D structure that has been implicated in lipid scrambling, which the authors investigate in the absence and presence of Ca²⁺ using a combination of cryo-EM structure determination, mutagenesis and functional assays. Their closed-groove structure reveals a continuous file of lipid molecules around the catalytic groove region, providing a structural basis for lipid interaction with the protein. Additionally, the authors capture three novel states of TMEM16, completing the picture of conformational transitions that this protein undergoes. Strikingly, the authors show that both structure and distribution of the protein’s conformations depend on lipid composition and nanodisc scaffold protein.

   Biophysics Colab considers this to be exceptional work and recommends it to scientists interested in plasma membrane lipid homeostasis and cryoEM.

   (This evaluation by Biophysics Colab refers to version 2 of this preprint, which has been revised in response to peer review of version 1.)

   Read the original source
2. 

   Biophysics Colab

   Feb 22, 2024

   Authors’ response (3 January 2024)

   GENERAL ASSESSMENT

   The TMEM16 protein family is composed of ten members in mammals, and fewer in lower eukaryotes. Members within this protein family play remarkably different roles: some serve as Ca²⁺-activated ion channels, others work as lipid scramblases in a Ca²⁺-dependent manner, and some combine the two functions. The molecular determinants responsible for lipid transport in TMEM16 scramblases are not fully defined. The current view of lipid scrambling is that, in presence of Ca²⁺, TMEM16 scramblases change their conformation to expose a hydrophilic ‘groove’ to the membrane. This destabilizes the lipid bilayer, enabling translocation of lipids (e.g. phosphatidylserine) from the inner to outer leaflet of the membrane. However, recent evidence suggests that scrambling can occur even when the …

   Authors’ response (3 January 2024)

   GENERAL ASSESSMENT

   The TMEM16 protein family is composed of ten members in mammals, and fewer in lower eukaryotes. Members within this protein family play remarkably different roles: some serve as Ca²⁺-activated ion channels, others work as lipid scramblases in a Ca²⁺-dependent manner, and some combine the two functions. The molecular determinants responsible for lipid transport in TMEM16 scramblases are not fully defined. The current view of lipid scrambling is that, in presence of Ca²⁺, TMEM16 scramblases change their conformation to expose a hydrophilic ‘groove’ to the membrane. This destabilizes the lipid bilayer, enabling translocation of lipids (e.g. phosphatidylserine) from the inner to outer leaflet of the membrane. However, recent evidence suggests that scrambling can occur even when the hydrophilic groove is closed.

   The new study by Feng and colleagues aims to investigate the molecular basis of closed-groove scrambling using the fungal scramblase, nhTMEM16. This protein was previously reported to maintain closed groove conformations even in the presence of Ca²⁺. The authors resolved a series of WT nhTMEM16 structures in two different nanodisc scaffolds, as well as several mutants with impaired scrambling. Strikingly, the conformational landscape of nhTMEM16 was found to rely on the lipid composition and scaffold used: the smaller E3D1 scaffold favored closed groove states and the larger 2N2 scaffold permitted intermediate and open-groove conformations. A high-resolution closed-groove structure obtained in E3D1 allowed the identification of a continuous file of lipid molecules around the catalytic groove region, providing a structural basis for lipid interaction with the closed groove. This complements prior work from this group involving a closely-related homolog, afTMEM16, in which the authors were able to visualize lipid molecules around the open groove. Furthermore, the authors succeeded in capturing three novel states of nhTMEM16 (Ca²⁺-free closed, Ca²⁺-bound intermediate-open and Ca²⁺-bound wider open states), completing the picture of conformational transitions that this protein undergoes upon activation.

   Mutation of key residues interacting with outer leaflet lipids selectively impaired scrambling in the absence of Ca²⁺. Residues involved in groove opening (E313-R432) were also identified and a mutation at this site (R432A) locked the nhTMEM16 scramblase in a closed-groove conformation, providing new insights into residues critical for groove opening. Furthermore, the authors tested the activity of nhTMEM16 mutants in several lipid compositions and reported striking differences, clarifying discrepancies from the authors’ prior work on nhTMEM16 using different lipid compositions and consolidating some of the observations from other TMEM16 homologs. It is noteworthy that the authors probed the effect of nanodisc size and lipid composition on nhTMEM16 conformation, providing thought-provoking insights for the membrane protein field. This approach is particularly valuable for closed-groove mutant structures, to ensure that the observed conformation is not dictated by scaffold size.

   Overall, this is a piece of carefully executed experimental work. The results are interpreted carefully in the context of the published literature, and the work provides important insight into plasma membrane lipid homeostasis. While the study does not have technical weaknesses, it could be improved in its presentation in order to make it more accessible to readers who are not experts in the TMEM16 field.

   We wish to thank the Colab editor and reviewers for their insightful comments, helpful suggestions, and appreciation of our work. We have extensively revised our manuscript to address their comments and suggestions. Below is a detailed point-by-point response to their suggestions.

   RECOMMENDATIONS

   Essential revisions:

   1. For readers not familiar with the field, some technical details might need to be explained in greater detail. For example:

   - In the section “Residues coordinating outer leaflet lipids are important in closed groove scrambling”, please indicate the method of measuring scrambling (liposome-based activity assay etc.) and refer to some of your prior work where the method is described for readers not familiar with the TMEM16 field. Additionally, it needs to be stated clearly what is considered a significant change in scrambling, as liposome assays are usually quite variable.

   We thank the reviewers for this suggestion. We edited the text to indicate the use of the well-established in vitro assay and added the relevant references (Lines 236-238).

   We illustrate the reproducibility of the experimental results by reporting in the bar charts the mean ± St. Dev of the scrambling rate constants, and by showing the values obtained from individual experiments (red dots superimposed to the bar charts). Additionally, we evaluated the statistical significance of the reported changes using Student’s t-test with Bonferroni correction. Finally, we added text discussing the limitations of our assay in lines 318-322.

   - Since prior work done by the group indicates that membrane thinning is a determinant of scrambling, and an open groove further thins the membrane to potentiate scrambling, it is not intuitive why the R432A mutant scrambles with WT-like rates in the presence of Ca²⁺. If this is due to the limitation of the assay (e.g. rate of NBD lipids bleaching), this should be stated more explicitly. Do the authors have insights from their structures regarding membrane thinning by R432A with/without Ca²⁺ and how that compares to WT protein?

   We thank the reviewers for raising this important point. In the presence of Ca2+ the fluorescence decay of N-NBD-PE in nhTMEM16 vesicles occurs with kinetics that are slightly slower than those of the chemical reduction step by dithionite. Therefore, while we can resolve two exponential components, it is possible we are underestimating the scrambling rate constants α and β. However, we note that a large slowing effect would be well resolved in our experimental conditions. In contrast, in the absence of Ca2+, which is the focus of our current analyses, scrambling is much slower than the chemical step and is well resolved. Finally, we note that the triple mutant Y327A/F330A/Y439A alone has no effect on scrambling in 0.5 mM Ca2+ but induces a ~8 fold reduction in the scrambling rate constants in 0 Ca2+. When this mutant is combined with R432A, which favors the closed groove conformation, we now see in the presence of Ca2+ the same ~8-fold reduction in the scrambling rate constants. This suggests that our assay can resolve effects even in the presence of Ca2+. This is discussed in Lines 318-322.

   We only determined the structure of R432A in the presence of Ca2+, therefore we cannot evaluate how Ca2+ binding affects membrane thinning in this mutant.

   - It is difficult to follow the reasoning for the R432A+Y327A/F330A/Y439A mutant phenotype. Is the assumption that Y327A/F330A/Y439A is in the open conformation with Ca²⁺, and therefore adding a mutation stabilizing the closed groove impairs scrambling in presence of Ca²⁺?

   We have expanded the rationale for this experiment in lines 307-315.

   - What the authors believe about the lipid pathway when the groove is open should be discussed in more detail and with reference to Alvadia et al 2019.

   We thank the reviewers for this important suggestion. We now explicitly state that: “With a closed groove, thinning is less pronounced, and scrambling is slower than when the groove is open, rationalizing the Ca2+ dependence of this process (Extended Data Fig. 10d-f).” (Lines 432-434) Since the present work is focused on the mechanism of closed groove scrambling, we prefer to refrain from adding more speculations on what happens when the groove is open, especially since this topic was the focus of a paper we recently published (Falzone, Feng et al., Nat Comms, 2022).

   2. A more detailed account of the physiological significance of the findings should be presented in the Discussion to offer reader the authors’ view on the broader implications of the work. Relevant points include:

   - Do the authors believe that conformational bias in nhTMEM16 in various cryo-EM conditions may be reflective of physiological regulation? Is it likely to happen in cells in vivo?

   This is an excellent point. We do hypothesize that the various observed conformation are physiological and indeed we explicitly state “…that the 7 observed conformations represent intermediates along the transition from apo closed to Ca2+ bound open” (Lines 444-445). Beyond this, we cannot speculate on whether the environmental dependent bias on nhTMEM16 can happen in a physiological context. We imagine that subtle changes in membrane composition can affect TMEM16 function, and indeed we see quite dramatic effects of lipid composition of scrambling activity, however whether these changes are reflective of shifts in the conformational landscape of groove opening, of effects of membrane properties, or both, it remains to be seen. Gaining definitive insights into this would require extensive additional structural experiments in unbounded membranes (i.e., from reconstituted liposomes of different composition or native vesicles, cell membranes) that are outside of the scope of the present work.

   - Do the authors believe that such regulation may also apply to mammalian TMEM16 scramblases or even channels?

   We consider this is a definite possibility, and now added a sentence stating that “This raises the possibility that unbounded membranes, such as those of liposomes, might perturb less the conformational landscape of the imaged proteins.” (Lines 499-501) However, without direct evidence we prefer to avoid speculating on this fascinating topic.

   - What implications do these findings have for our understanding of lipid scrambling mechanisms by TMEM16 scramblases that work in intracellular (thinner) membranes (such as TMEM16K)?

   We agree this is an important point. We now added a sentence stating “The strong dependence of closed groove scrambling on membrane properties could provide a mode of regulation of TMEM16 activity in cellular membranes, such as the cholesterol rich plasma membrane or the thinner ER membrane.” (Lines 434-436)

   - What implications might the knowledge of residues involved in lipid scrambling of closed scramblases potentially have for medicine and therapy? Can the authors speculate as to whether the identified residues have the potential to be tackled pharmacologically and what use could this have?

   We do not know whether the residues we identified as important for closed groove scrambling could provide a pathway to pharmacological manipulation of TMEM16 scramblase activity. This is a fascinating topic, especially in light of the very poor availability of pharmacological tools to manipulate TMEM16 scramblase activity. However, at present it remains speculative and outside the scope of the present manuscript.

   More generally, what is the physiological role of lipid transport in the absence of Ca²⁺? Does this constitute a lipid "leak”?

   This is an excellent question. One possibility is that scramblases have a basal activity, that in cellular homeostasis is counteracted by the activity of flippases and floppases. Alternatively (or complementarily), it is possible that in the context of an unperturbed native membrane the basal activity is negligible. However, we do not have data addressing the present point and therefore our hypotheses remain limited to pure speculations, therefore we prefer to maintain the focus of the present manuscript on the mechanism of closed groove scrambling and on the potential effects that the environment can have on the interpretation cryoEM imaging experiments.

   Optional suggestions:

   1. Regarding residues involved in groove opening (E313-R432), it would be very interesting to expand the work by studying additional mutants and investigating more fully the role of E313 in DOPC:DOPG lipids, since at present only a mutation in R432 was tested experimentally in this lipid composition.

   We agree with the reviewers that expanding the analysis to other residues, such as E313, would be interesting. However, initial functional experiments suggested this mutant behaves similarly to R432A, and thus we did not think it would provide much additional mechanistically insights to what we already have.

   2. Measurements of ion transport in nhTMEM16 would also be useful to further validate the closed groove conformation of R432A. This could shed new light onto whether ion transport and lipid transport are coupled in TMEM16 proteins.

   This is an excellent suggestion, one that indeed we considered at length during this project. Ultimately, we decided not to pursue this avenue of investigations because of the limitations of the flux assay for non-specific ion channels. While flux assays can provide quantitative measures of effects for anion or cation selective channels, for non-selective channels these assays only provide very coarse yes/no answers (i.e., whether the construct mediates any channel activity or not). Since we expected these mutants might have intermediate phenotypes, rather than completely ablating channel activity, we were concerned that the experiments would be inconclusive at best or, at worst, misleading. These limitations are extensively discussed in our previous manuscripts (Lee et al., Nat Comms, 2018; Falzone and Accardi, Methods Mol Biol, 2020).

   3. Since the authors found significant differences in their new structures with previously reported, how do Ca²⁺-bound closed structures of nhTMEM16 in POPC/POPG (previously published) and DOPC/DOPG (obtained in this study) compare to each other?

   We thank the reviewers for this suggestion. In Lines 167-168 we now state: “The Ca2+ bound closed conformations in MSP1E3 DOPC/DOPG (PDBID: 6QMB) and MSP2N2 POPC/POPG are nearly identical (Cα r.m.s.d ~0.50 Å).”

   4. The purpose of creating composite symmetric maps from symmetry expanded monomers is questionable – if it is not possible to isolate this symmetric state by classification approaches, it is probably very transient, or not present at all. However, there are no strict guidelines, and it is acceptable as long as everything is described in MM and all the maps deposited. Are composite and monomer E3D1 apo maps deposited alongside the main map as EMD-41477?

   We agree with the reviewers that depositing the maps of the unexpanded dimers is appropriate and opportune, and indeed we did so

   i. the combined dimer map which was primarily used for model building is deposited as EMDB: 41453 and the model as PDB: 8TOI;

   ii. the local refined monomer map was deposited as EMDB: 41458

   iii. the dimer consensus map used for map combination was deposited as EMDB: 41457

   The rationale to generate a combined dimer map is that this allows for a better visualization of the protein-bilayer interface and the ensuing distortions. When viewing the map of a single monomer it is difficult to appreciate these effects.

   5. The authors show that Ca²⁺-dependent α6 straightening is important for closed-groove scrambling. This is directly relevant for TMEM16F, for which this is the only conformational change observed. The authors note that extracellular α4 is more mobile in R432A mutant, is this in any way similar to the conformations reported for more active TMEM16F mutants (Arndt et al., 2022)?

   What we see is that the density for the top of TM4 becomes very weak. This is quite different from what Arndt et al. reported, where they see a significant and defined movement of both TM4 and TM3. While we think many of the basic mechanisms of closed-groove scrambling we and many others are beginning to unravel are likely conserved across TMEM16 homologues, it is very likely that differences will exist between homologues. We now make this important point in Lines 432-434.

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

   Read the original source
3. 

   Biophysics Colab

   Feb 22, 2024

   Consolidated peer review report (11 October 2023)

   GENERAL ASSESSMENT

   The TMEM16 protein family is composed of ten members in mammals, and fewer in lower eukaryotes. Members within this protein family play remarkably different roles: some serve as Ca²⁺-activated ion channels, others work as lipid scramblases in a Ca²⁺-dependent manner, and some combine the two functions. The molecular determinants responsible for lipid transport in TMEM16 scramblases are not fully defined. The current view of lipid scrambling is that, in presence of Ca²⁺, TMEM16 scramblases change their conformation to expose a hydrophilic ‘groove’ to the membrane. This destabilizes the lipid bilayer, enabling translocation of lipids (e.g. phosphatidylserine) from the inner to outer leaflet of the membrane. However, recent evidence suggests that scrambling can occur even …

   Consolidated peer review report (11 October 2023)

   GENERAL ASSESSMENT

   The TMEM16 protein family is composed of ten members in mammals, and fewer in lower eukaryotes. Members within this protein family play remarkably different roles: some serve as Ca²⁺-activated ion channels, others work as lipid scramblases in a Ca²⁺-dependent manner, and some combine the two functions. The molecular determinants responsible for lipid transport in TMEM16 scramblases are not fully defined. The current view of lipid scrambling is that, in presence of Ca²⁺, TMEM16 scramblases change their conformation to expose a hydrophilic ‘groove’ to the membrane. This destabilizes the lipid bilayer, enabling translocation of lipids (e.g. phosphatidylserine) from the inner to outer leaflet of the membrane. However, recent evidence suggests that scrambling can occur even when the hydrophilic groove is closed.

   The new study by Feng and colleagues aims to investigate the molecular basis of closed-groove scrambling using the fungal scramblase, nhTMEM16. This protein was previously reported to maintain closed groove conformations even in the presence of Ca²⁺. The authors resolved a series of WT nhTMEM16 structures in two different nanodisc scaffolds, as well as several mutants with impaired scrambling. Strikingly, the conformational landscape of nhTMEM16 was found to rely on the lipid composition and scaffold used: the smaller E3D1 scaffold favored closed groove states and the larger 2N2 scaffold permitted intermediate and open-groove conformations. A high-resolution closed-groove structure obtained in E3D1 allowed the identification of a continuous file of lipid molecules around the catalytic groove region, providing a structural basis for lipid interaction with the closed groove. This complements prior work from this group involving a closely-related homolog, afTMEM16, in which the authors were able to visualize lipid molecules around the open groove. Furthermore, the authors succeeded in capturing three novel states of nhTMEM16 (Ca²⁺-free closed, Ca²⁺-bound intermediate-open and Ca²⁺-bound wider open states), completing the picture of conformational transitions that this protein undergoes upon activation.

   Mutation of key residues interacting with outer leaflet lipids selectively impaired scrambling in the absence of Ca²⁺. Residues involved in groove opening (E313-R432) were also identified and a mutation at this site (R432A) locked the nhTMEM16 scramblase in a closed-groove conformation, providing new insights into residues critical for groove opening. Furthermore, the authors tested the activity of nhTMEM16 mutants in several lipid compositions and reported striking differences, clarifying discrepancies from the authors’ prior work on nhTMEM16 using different lipid compositions and consolidating some of the observations from other TMEM16 homologs. It is noteworthy that the authors probed the effect of nanodisc size and lipid composition on nhTMEM16 conformation, providing thought-provoking insights for the membrane protein field. This approach is particularly valuable for closed-groove mutant structures, to ensure that the observed conformation is not dictated by scaffold size.

   Overall, this is a piece of carefully executed experimental work. The results are interpreted carefully in the context of the published literature, and the work provides important insight into plasma membrane lipid homeostasis. While the study does not have technical weaknesses, it could be improved in its presentation in order to make it more accessible to readers who are not experts in the TMEM16 field.

   RECOMMENDATIONS

   Essential revisions:

   1. For readers not familiar with the field, some technical details might need to be explained in greater detail. For example:

   - In the section “Residues coordinating outer leaflet lipids are important in closed groove scrambling”, please indicate the method of measuring scrambling (liposome-based activity assay etc.) and refer to some of your prior work where the method is described for readers not familiar with the TMEM16 field. Additionally, it needs to be stated clearly what is considered a significant change in scrambling, as liposome assays are usually quite variable.

   - Since prior work done by the group indicates that membrane thinning is a determinant of scrambling, and an open groove further thins the membrane to potentiate scrambling, it is not intuitive why the R432A mutant scrambles with WT-like rates in the presence of Ca²⁺. If this is due to the limitation of the assay (e.g. rate of NBD lipids bleaching), this should be stated more explicitly. Do the authors have insights from their structures regarding membrane thinning by R432A with/without Ca²⁺ and how that compares to WT protein?

   - It is difficult to follow the reasoning for the R432A+Y327A/F330A/Y439A mutant phenotype. Is the assumption that Y327A/F330A/Y439A is in the open conformation with Ca²⁺, and therefore adding a mutation stabilizing the closed groove impairs scrambling in presence of Ca²⁺?

   - What the authors believe about the lipid pathway when the groove is open should be discussed in more detail and with reference to Alvadia et al 2019.

   2. A more detailed account of the physiological significance of the findings should be presented in the Discussion to offer reader the authors’ view on the broader implications of the work. Relevant points include:

   - Do the authors believe that conformational bias in nhTMEM16 in various cryo-EM conditions may be reflective of physiological regulation? Is it likely to happen in cells in vivo?

   - Do the authors believe that such regulation may also apply to mammalian TMEM16 scramblases or even channels?

   - What implications do these findings have for our understanding of lipid scrambling mechanisms by TMEM16 scramblases that work in intracellular (thinner) membranes (such as TMEM16K)?

   - What implications might the knowledge of residues involved in lipid scrambling of closed scramblases potentially have for medicine and therapy? Can the authors speculate as to whether the identified residues have the potential to be tackled pharmacologically and what use could this have? More generally, what is the physiological role of lipid transport in the absence of Ca²⁺? Does this constitute a lipid "leak”?

   Optional suggestions:

   1. Regarding residues involved in groove opening (E313-R432), it would be very interesting to expand the work by studying additional mutants and investigating more fully the role of E313 in DOPC:DOPG lipids, since at present only a mutation in R432 was tested experimentally in this lipid composition.

   2. Measurements of ion transport in nhTMEM16 would also be useful to further validate the closed groove conformation of R432A. This could shed new light onto whether ion transport and lipid transport are coupled in TMEM16 proteins.

   3. Since the authors found significant differences in their new structures with previously reported, how do Ca²⁺-bound closed structures of nhTMEM16 in POPC/POPG (previously published) and DOPC/DOPG (obtained in this study) compare to each other?

   4. The purpose of creating composite symmetric maps from symmetry expanded monomers is questionable – if it is not possible to isolate this symmetric state by classification approaches, it is probably very transient, or not present at all. However, there are no strict guidelines, and it is acceptable as long as everything is described in MM and all the maps deposited. Are composite and monomer E3D1 apo maps deposited alongside the main map as EMD-41477?

   5. The authors show that Ca²⁺-dependent α6 straightening is important for closed-groove scrambling. This is directly relevant for TMEM16F, for which this is the only conformational change observed. The authors note that extracellular α4 is more mobile in R432A mutant, is this in any way similar to the conformations reported for more active TMEM16F mutants (Arndt et al., 2022)?

   REVIEWING TEAM

   Reviewed by:

   Anna Boccaccio, Senior Research Scientist, Istituto di Biofisica, Italy: electrophysiology, biophysics of channels and scramblases

   Valeriia Kalienkova, Postdoctoral Researcher, University of Bergen, Norway: membrane proteins, single particle cryo-EM

   Paolo Tammaro, Professor, University of Oxford, UK: molecular and systems physiology and pharmacology of ion and lipid transport

   Curated by:

   Michael Pusch, Research Director, Istituto di Biofisica, Italy

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

   Read the original source
4. 

   Version published to 10.1101/2023.08.11.553029v2 on bioRxiv

   Jan 30, 2024
5. 

   Version published to 10.1101/2023.08.11.553029v1 on bioRxiv

   Aug 12, 2023