Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats

MORN (Membrane Occupation and Recognition Nexus) repeat proteins have a wide taxonomic distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain poorly characterised at both a structural and a functional level compared to other common repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate membrane targeting. We addressed this putative activity by focusing on a protein composed solely of MORN repeats—Trypanosoma brucei MORN1. Surprisingly, no evidence for binding to membranes or lipid vesicles by TbMORN1 could be obtained either in vivo or in vitro. Conversely, TbMORN1 did interact with individual phospholipids. High- and low-resolution structures of the MORN1 protein from Trypanosoma brucei and homologous proteins from the parasites Toxoplasma gondii and Plasmodium falciparum were obtained using a combination of macromolecular crystallography, small-angle X-ray scattering, and electron microscopy. This enabled a first structure-based definition of the MORN repeat itself. Furthermore, all three structures dimerised via their C-termini in an antiparallel configuration. The dimers could form extended or V-shaped quaternary structures depending on the presence of specific interface residues. This work provides a new perspective on MORN repeats, showing that they are protein-protein interaction modules capable of mediating both dimerisation and oligomerisation.


Summary of main points by the reviewers, and authors' responses:
• Both reviewers felt that the manuscript was overlong; Reviewer 1 recommended either shortening it or splitting it into two stories, while Reviewer 2 recommended cutting down the text.
o We have considerably shortened the manuscript in accordance with this request (see revision plan below). We had already considered splitting the manuscript into two parts during the drafting stage, and had rejected this possibility as the data are intertwined -the retroactive validation of the dimer interface by the mutagenesis constructs (OV Fig. S3 [NV Fig. S4]) being a good example.
o The revised manuscript features 7 main figures and 13 supplementals. • Both reviewers felt too much text and figure space was allocated to negative data, specifically the investigation of potential lipid binding by the TbMORN1 protein, and that there should be more focus on the positive parts of the story. o A key part of shortening the manuscript has been moving most of the negative data on lipid binding into the supplemental figures, and considerably shortening the associated text. This has allowed the main figures and associated text to focus more on the positive elements of the project, while still ensuring publication of all the data. • The reviewers appear to be in slight disagreement concerning discussion of the data.
Reviewer 1 has encouraged more speculation on the physiological role of PE binding, a potential lipid transfer function, a role for calcium ions, the relevance of the observed disulphide bond, and the role of zinc ions in apicomplexan proteins; Reviewer 2 has recommended avoiding excessive speculation or inference.
o Given that both reviewers have agreed that the original manuscript was overlong, we have implemented Reviewer 2's suggestion here and reduced the amount of speculation in the revised text. • The reviewers agreed that the technical quality of the data was high and that the conclusions drawn were robust.
o We are glad that the reviewers were appreciative of the data quality. For this reason, we were reluctant to remove any of the data from the manuscript and would prefer instead to transfer it to the supplementals. We feel that the negative data still have considerable community value, given that they show that MORN repeats are not automatically lipid-binding modules and can thus act as a caveat to other researchers.
2. Detailed revision plan for the preprint: • We have implemented the reviewers' suggestions and substantially shortened the manuscript, primarily by trimming the (phospho)lipid-binding section, which contains a large amount of negative data. The following main figures have been moved into the supplemental section: ▪ OV Fig. 2 ("TbMORN1 interacts with phospholipids but not liposomes") has become NV Fig o We have additionally limited the amount of inference and speculation in the manuscript.
3. Point-by-point responses to the reviewers The authors' responses are shown in red italics.
Reviewer #1 (Evidence, reproducibility and clarity (Required)): MORN (membrane occupation and recognition nexus) repeat proteins are found in prokaryotes and eukaryotes. They feature characteristic repeats in their primary sequence, have been assumed to play a role in lipid binding, but remain poorly characterized on the functional and structural level. This manuscript tries to address both these questions and is organized in major parts. In the first part the authors characterize a putative role of MORN repeat proteins in lipid binding and membrane association. In the second part, the authors use X-ray crystallography to establish the structure of MORN repeat proteins and to investigate the dimerization.
As a cleverly chosen point of departure, they focus their study particularly on MORN1 from Trypanosoma brucei (TbMORN1), which is composed solely on MORN repeats. The structures of MORN repeats (from several species) in part two provide interesting insights into their mode of homotypic interactions and their role as dimerization or oligomerization devices.
The lipid binding and membrane association of MORN proteins in the first part remains somewhat confusing and unclear, despite the use of a whole battery of techniques. We anticipate that the shortening and refocusing of the lipid binding data has addressed this issue.
It is questionably, why the authors invest so many figures and words to inform the reader on negative results. Again, our expectation is that the major changes planned for the data presentation in this section will make it more coherent.
The main issue of this manuscript is, in my view, the way the data were presented.The manuscript is generally well-written, but much too long. The structural work is important and concise.
We have considerably shortened the manuscript as per the reviewer's request, and especially the section on lipid binding. In the current form, the first part and the second part of the manuscript remain somewhat detached from each other. The characterization of the lipid binding/membrane binding properties has a number of substantial weaknesses (e.g. use of quite different, nonphysiological buffers for membrane binding assays; use of deletion mutants for the binding assays, which do not show the full potential of oligomerization). This which makes it hard to read and confuses the reader. Even though I have no reason to doubt the conclusions by the authors, I do not think that all necessary caution has been invested to rule out other possibilities.
We believe that the shortening and refocusing of the manuscript should address these issues.

For consideration of the buffer and deletion mutant points, please see responses to Major Points below.
In summary, even though the technical quality of the individual performed assays is high, there are some conceptual issues that make it hard to make a strong case based on a collection of individual, clear datasets. Even though I find the structures of the MORN proteins important, timely, and interesting, I would not recommend this study for publication in its current form. The manuscript would be more fun to read if both of the parts would be shortened substantially and more focused.
We have implemented this suggestion: the manuscript has been considerably shortened (from 20,489/135,073 to 18,555/103,988 characters/words, focused on reducing the negative lipidbinding results).
While I agree that most evidence provided on lipid/membrane binding of TbMORN1 argue against a direct role of MORN proteins in membrane binding, I feel that the experimental approach is not coherent enough. See a few major points of criticism below.

**Major Points:**
1) The authors decide to characterize the membrane binding of a MORN repeat protein using a deletion variant that lacks the N-terminal repeat. However, in Figure 1B they show that the N-terminal repeat is important for the formation of higher-order oligomers. While I fully understand that the presence of the most N-terminal repeat does hamper the structural work, I find it problematic to remove it for the lipid/membrane-binding assays. The formation of higher oligomeric species beyond the dimer, may be important for membrane binding/recruitment (avidity effects).
As we explained in the manuscript, the reason for not using the full-length protein for in vitro work was because it was polydisperse, and that the yields were extremely low. See OV lines 178-179 ("The yields of TbMORN1(1-15) were always very low, making this construct not generally suitable for in vitro assays".) and OV lines 411-414 ("...TbMORN1(1-15), which was polydisperse in vitro and formed large oligomers (Fig. 1B). The membrane-binding activity of these polydisperse oligomers was not possible to test in vitro, as the purification yields of TbMORN1(1-15) were always low."). Consequently, we used the longest construct that was suitable in terms of chemical and oligomeric homogeneity. Using the full-length protein would have had inherent problems with aggregation, and consequently would have compromised the data and derived results. In order to make this clear in the manuscript we edited the sentence mentioned above as follows:

"It was not possible to test the membrane-binding activity of these polydisperse oligomers in vitro however, as the purification yields of TbMORN1(1-15) were always low. As an alternative, the possible membrane association of TbMORN1(1-15) was examined in vivo."
2) (Related to point 1) I do not understand the choice of the buffers used for some of the assays. The use of pH 8.5 and NaCl concentrations of 200 mM are non-physiological. These were the buffer conditions required to retain the protein in a monodisperse state, suitable for in vitro assays.
For CD spectroscopy, a high ionic strength was obtained by the use of 200 mM NaF. If a high ionic strength is required to prevent the formation of higher oligomers of MORN, it raises the question if the formation of higher oligomers (under physiological conditions) may also contribute to their function. For example: In Figure 2E TbMORN(2-15) does show some concentration-dependent binding, which -however-is interpreted as background binding. What are the results using this assay (or better: a liposome floatation assay) when using full-length TbMORN(1-15) in a more physiological buffer? As noted already, it is not possible to use the TbMORN1(1-15) construct for in vitro assays owing to the extremely low yields and polydisperse nature of the protein. The excess fulllength protein was associated with the cytosolic fraction and not the membrane fraction in vivo (OV Fig. 6B [NV Fig. 4B]).
The statement that MORN proteins bind to lipids, but not to liposomes/membranes is -in my view-not sufficiently addressed to make a strong case. At no point do we suggest that MORN repeat proteins in general bind to lipids and not to liposomes/membranes. On the contrary, and as detailed in the manuscript, we set out to assay the lipid binding activity of TbMORN1, found that it appears to bind to lipids but not to liposomes/membranes, and have therefore cautioned that lipid or liposome/membrane binding of other MORN repeat proteins must be tested experimentally before claims of function are made.
3) The physiological relevance of lipid binding to MORN proteins remains obscure (as also acknowledged by the authors). Does the binding of PE lipids to the MORN protein have a physiological role? Does the binding of fluorescent PI(4,5)P2 point to a physiological role of MORN proteins?
These are interesting questions that we would like to address in future work.

4)
In light of recent data from the Chris Stefan lab (PMID: 31402097) a co-incidence detection of PI(4,5)P2, PS, and cholesterol seems possible. Can the authors address this possibility? Again, the involvement of cholesterol, PS, and PI(4,5)P2 would be interesting questions for subsequent work but are beyond the scope of the present study. We did partially address this issue in our use of PI(4,5)P2, POPC and cholesterol containing liposomes in liposome cosedimentation assays, which showed no binding (OV Fig. S3A [NV Fig. S4A]).
Furthermore, the role of Ca2+ signaling / Ca2+ ions has not been addressed. Despite my critique raised above, I agree with the authors that the data point in the direction that MORN proteins (or at least TbMORN1) does not directly bind to membranes. Their data, however, would still be consistent with a role as lipid transfer protein and a recruitment of MORN proteins to the membrane by other proteins. Have the authors performed any additional experiments in this direction? Also, the potential role of palmitoylation is only mentioned in the discussion (page 22), while palmitoylation would provide a simple means for membrane recruitment.
We are glad that the reviewer concurs with our main conclusion. We agree, as noted in the discussion, that a role as a lipid transfer protein might still be possible, and this is something that we would like to pursue in follow-up work. We have not yet performed any additional experiments in this direction. Concerning palmitoylation, the predictions using the CSS-Palm software were always weak and ambiguous, and in addition the best candidate cysteine residue was Cys351, which is in our structure engaged in the disulphide bond observed in the C2 crystal form. We feel that this is something to keep in mind, but is not yet a strong enough hypothesis to pursue intensively. Figure 1B: The authors should provide information on the void volume of the column. Implemented in the figure legend (7.2 ml).

Reviewer #1 (Significance (Required)):
A putative role of MORN proteins in membrane and lipid binding is addressed. The view the MORN proteins bind directly to membranes is challenged. Structures of dimeric MORN proteins provide important insight into the modes of dimerization.
There is a recent structure of MORN proteins (which is referenced by the authors), but I feel that additional structural work is important and justified. The work on membrane vs. lipid binding is important, but not sufficiently addressed in the current manuscript.
We are glad that the reviewer finds the structural work important and justified, although we disagree with the reviewer's assessment of the lipid binding. As noted in the previous paragraph, our data challenge the assumption that MORN repeat proteins directly bind membranes, and we feel that this alone is a significant conceptual advance.
I would recommend to separate the study in two parts. The audience is likely to confused (or bored) by the lengthy discussion on whether or not MORN proteins bind lipids and or membrane or not.
We would prefer to implement the reviewer's other suggestion, namely that the manuscript is considerably shortened and less focus given to the negative data on lipid binding.
I am not an expert in structural biology, but have a fair understanding of structural biology. I have worked on lipid binding proteins and have a very good understanding of lipid/membrane-binding assays.

**Summary**
The manuscript describes an extensive and detailed investigation into the structure and function(s) of MORN domains. It has to be acknowledged that, despite the considerable amount of work reported, the conclusions are rather limited. From a technical viewpoint, the experiments have been appropriately executed and, generally, I concur with the conclusions drawn. However, the manuscript is over-long: in general, I would recommend concentrating on positive conclusions which can be drawn from the data and avoid excessive speculation or inference (some examples given below).
We are glad that the reviewer is satisfied with the technical quality of the work and (in general) the validity of the conclusions. We acknowledge that the original submission was fairly long, and have considerably shortened the revised manuscript and focused more on the positive conclusions in order to implement this suggestion.

**Major Comments**
There are three general-perhaps rather obvious-points to make. First, there is no particular reason to think that conservation of structure necessarily indicates conservation of a particular function. There seems to be an implicit assumption that MORN domains are associated with a specific, well-defined biological function. Given their diversity, are there particular reasons to think that this is the case?
The reviewer is exactly right that there is an implicit assumption that MORN domains are associated with a specific, well-defined function: specifically, lipid binding. It is this assumption, which has been widely circulated in the almost complete absence of experimental evidence, that we are challenging. We agree that MORN repeats are likely to be capable of multiple functions, and protein-protein interactions are now better supported than protein-lipid interactions.
Second, a strategy which examines the properties of just the recombinant MORN domains in vitro, removed from the context of the whole protein (eg junctophilin) or-importantly-its interacting partners in vivo, has obvious limitations. Frequently a reductionist approach is successful; however, in this case, MORN domains appear to be less tractable to that kind of approach. For all the in vitro binding and structural experiments presented, there is always a concern that the absence of other parts of the relevant MORN-containing protein or its partners could explain failure or inconsistency of in vitro biological activity measurements. Again, the reviewer is right that there is an inherent contextual limitation to any in vitro work that utilises a single protein, but this is a concern that -by definition -could be raised about any in vitro study utilising a single protein. It (Fig. 7G) 3. Fig 2C: did the authors also estimate binding stoichiometry as well as the equilibrium binding constants for these data? This should be determined by fitting a single binding site model to the data. Other methods (eg ITC) can probably determine this with more accuracy. The value of stoichiometry is sometimes forgotten in such binding measurements-is one ligand bound per monomer or dimer, for example? We discussed estimation of the binding stoichiometry in the fluorescence anisotropy assays at some length, but the conclusion was that the required experiments would contain too many approximations to provide high-confidence data. We did use ITC and also MST, but did not observe any binding with these assays.
4. Lines 674-678 I found it hard to work out whether these constructs harbour the natural Cterminal sequence without truncation or addition of an affinity tag. I think the answer is 'yes' but it was difficult working this out from the details in M&M. TbMORN1(7-15) crystallisation was with a C-terminal Strep tag; TgMORN1(7-15) and PfMORN1(7-15) had their affinity tags removed by protease treatment prior to crystallisation. We have clarified this point in the M&M, page 29, lines 1189-1192: "Crystallisation of TbMORN1(7

-15) (with a C-terminal Strep tag), TgMORN1(7-15) and PfMORN1(7-15) (both with affinity tags removed) was performed at 22 °C using a sitting-drop vapour diffusion technique and micro-dispensing liquid handling robots (Phoenix RE (Art Robbins Instruments) and Mosquito (TTP labtech)."
5. Lines 688-694 The PISA interface analysis is useful here in distinguishing crystal contacts from those which persist in solution. The discussion of the results is unclear, however, on this critical point: were the dimer interfaces the only contacts which were significant in the various crystal forms? Yes, correct. PISA showed that the described dimerisation contacts were the only significant ones in the various crystal forms. Other crystals contacts had typically low P-values and poor ΔG and small "radar" surface in the complexive PISA analysis.
In the case of both TbMORN1 crystal forms and in the case of the TgMORN1 P43212 crystal form we have a dimer in the asymmetric unit, while in the case of the PfMORN1 and TgMORN1 P6222 form we have one molecule in the asymmetric unit, and the dimer is created by the crystallographic twofold axis. In the latter cases the quaternary structure resulting from the symmetry operations was the top-scoring one considering either P-values and/or the number of stabilising interactions buried surface area.
6. Lines 754-763 This paragraph seems rather speculative and is a good example where the text could be cut down.
If the line citation is correct, then we disagree with this assessment and would prefer not to implement it. The paragraph in question concerns a detailed and very precise discussion of the side chain interactions that stabilise the V-shaped forms of TgMORN1 and PfMORN1.
7. Line 765-788 This section is also rather overdone: such observations are only useful if they are subsequently tested by recording dimer conformation for a representative selection of MORN dimers from different species. Again, we disagree with the reviewer's assessment of this analysis. The analysis has considerable predictive power and already has some experimental validation via the SAXS observation that PfMORN1 is capable of forming extended dimers in solution (OV Fig. 10C [NV Fig. 7C]).