Structural basis of closed groove scrambling by a TMEM16 protein

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.)