Protein-Induced Membrane Strain Drives Supercomplex Formation

Reviewer #1 (Public review):

This paper by Poverlein et al reports the substantial membrane deformation around the oxidative phosphorylation super complex, proposing that this deformation is a key part of super complex formation. I found the paper interesting and well-written but identified a number of technical issues that I suggest should be addressed:

(1) Neither the acyl chain chemical makeup nor the protonation state of CDL are specified. The acyl chain is likely 18:2/18:2/18:2/18:2, but the choice of the protonation state is not straightforward.

(2) The analysis of the bilayer deformation lacks membrane mechanical expertise. Here I am not ridiculing the authors - the presentation is very conservative: they find a deformed bilayer, do not say what the energy is, but rather try a range of energies in their Monte Carlo model - a good strategy for a group that focuses on protein simulations. The bending modulus and area compressibility modulus are part of the standard model for quantifying the energy of a deformed membrane. I suppose in theory these might be computed by looking at the per-lipid distribution in thickness fluctuations, but this route is extremely perilous on a per-molecule basis. Instead, the fluctuation in the projected area of a lipid patch is used to imply the modulus [see Venable et al "Mechanical properties of lipid bilayers from molecular dynamics simulation" 2015 and citations within]. Variations in the local thickness of the membrane imply local variations of the leaflet normal vector (the vector perpendicular to the leaflet surface), which is curvature. With curvature and thickness, the deformation energy is analyzed.

See:
Two papers: "Gramicidin A Channel Formation Induces Local Lipid Redistribution" by Olaf Andersen and colleagues. Here the formation of a short peptide dimer is experimentally linked to hydrophobic mismatch. The presence of a short lipid reduces the influence of the mismatch. See below regarding their model cardiolipin, which they claim is shorter than the surrounding lipid matrix.

Also, see:
Faraldo-Gomez lab "Membrane transporter dimerization driven by differential lipid solvation energetics of dissociated and associated states", 2021. Mondal et al "Membrane Driven Spatial Organization of GPCRs" 2013 and many citations within these papers.

While I strongly recommend putting the membrane deformation into standard model terms, I believe the authors should retain the basic conservative approach that the membrane is strongly deformed around the proteins and that making the SC reduces the deformation, then exploring the consequences with their discrete model.

(1) If CDL matches the hydrophobic thickness of the protein it would disrupt SC formation, not favor it. The authors' hypothesis is that the SC stabilizes the deformed membrane around the separated elements. Lipids that are compatible with the monomer deformed region stabilize the monomer, similarly to a surfactant. That is, if CDL prefers the interface because the interface is thin and their CDL is thin, CDL should prevent SC formation. A simpler hypothesis is that CDL's unique electrostatics are part of the glue.

(2) Error bars for lipid and Q* enrichments should be computed averaging over multi-lipid regions of the protein interface, e.g., dividing the protein-lipid interface into six to ten domains, in particular functionally relevant regions. Anionic lipids may have long, >500 ns residence times, which makes lipid enrichment large and characterization of error bars challenging in short simulations. Smaller regions will be noisy. The plots depicted in, for example, Figure S2 are noisy.

(3) The membrane deformation is repeatedly referred to as "entropic" without justification. The bilayer has significant entropic and enthalpic terms just like any biomolecule, why are the authors singling out entropy? The standard "Helfrich" energetic Hamiltonian is a free energy model in that it implicitly integrates over many lipid degrees of freedom.

(4) Figure S7 shows the surface area per lipid and leaflet height. This appears to show a result that is central to the interpretation of SC formation but which makes very little sense. One simply does not increase both the height and area of a lipid. This is a change in the lipid volume! The bulk compressibility of most anything is much higher than its Young's modulus [similar to area compressibility]. Instead, something else has happened. My guess is that there is *bilayer* curvature around these proteins and that it has been misinterpreted as area/thickness changes with opposite signs of the two leaflets. If a leaflet gets thin, its area expands. If the manuscript had more details regarding how they computed thickness I could help more. Perhaps they measured the height of a specific atom of the lipid above the average mid-plane normal? The mid-plane of a highly curved membrane would deflect from zero locally and could be misinterpreted as a thickness change.

(5) The authors write expertly about how conformational changes are interpreted in terms of function but the language is repeatedly suggestive. Can they put their findings into a more quantitative form with statistical analysis? "The EDA thus suggests that the dynamics of CI and CIII2 are allosterically coupled."

(6) The authors write "We find that an increase in the lipid tail length decreases the relative stability of the SC (Figure S5C)" This is a critical point but I could not interpret Figure S5C consistently with this sentence. Can the authors explain this?

(7) The authors use a 6x6 and 15x15 lattice to analyze SC formation. The SC assembly has 6 units of E_strain favoring its assembly, which they take up to 4 kT. At 3 kT, the SC should be favored by 18 kT, or a Boltzmann factor of 10^8. With only 225 sites, specific and non-specific complex formation should be robust. Can the authors please check their numbers or provide a qualitative guide to the data that would make clear what I'm missing?

In summary, the qualitative data presented are interesting (especially the combination of molecular modeling with simpler Monte Carlo modeling aiding broader interpretation of the results) ... but confusing in terms of the non-standard presentation of membrane mechanics and the difficulty of this reviewer to interpret some of the underlying figures: especially, the thickness of the leaflets around the protein and the relative thickness of cardiolipin. Resolving the quantitative interpretation of the bilayer deformation would greatly enhance the significance of their Monte Carlo model of SC formation.

Reviewer #2 (Public review):

Summary:

The authors have used large-scale atomistic and coarse-grained molecular dynamics simulations on the respiratory chain complex and investigated the effect of the complex on the inner mitochondrial membrane. They have also used a simple phenomenological model to establish that the super complex (SC) assembly and stabilisation are driven by the interplay between the "entropic" forces due to strain energy and the enthalpies forces (specific and non-specific) between lipid and protein domains. The authors also show that the SC in the membrane leads to thinning and there is preferential localisation of certain lipids (Cardiolipin) in the annular region of the complex. The data reports that the SC assembly has an effect on the conformational dynamics of individual proteins making up the assembled complex and they undergo "allosteric crosstalk" to maintain the stable functional complex. From their conformational analyses of the proteins (individual and while in the complex) and membrane "structural" properties (such as thinning/lateral organization etc) as well from the out of their phenomenological lattice model, the authors have provided possible implications and molecular origin about the function of the complex in terms of aspects such as charge currents in internal mitochondrion membrane, proton transport activity and ATP synthesis.

Strengths:

The work is bold in terms of undertaking modelling and simulation of such a large complex that requires simulations of about a million atoms for long time scales. This requires technical acumen and resources. Also, the effort to make connections to experimental readouts has to be appreciated (though it is difficult to connect functional pathways with limited (additive forcefield) simulations.

Weakness:

There are several weaknesses in the paper (please see the list below). Claims such as "entropic effect", "membrane strain energy" and "allosteric cross talks" are not properly supported by evidence and seem far-fetched at times. There are other weaknesses as well. Please see the list below.

(i) Membrane "strain energy" has been loosely used and no effort is made to explain what the authors mean by the term and how they would quantify it. If the membrane is simulated in stress-free conditions, where are strains building up from?

(ii) In result #1 (Protein membrane interaction modulates the lipid dynamics ....), I strongly feel that the readouts from simulations are overinterpreted. Membrane lateral organization in terms of lipids having preferential localisation is not new (see doi: 10.1021/acscentsci.8b00143) nor membrane thinning and implications to function (https://doi.org/10.1091/mbc.E20-12-0794). The distortions that are visible could be due to a mismatch in the number of lipids that need to be there between the upper and lower leaflets after the protein complex is incorporated. Also, the physiological membrane will have several chemically different lipids that will minimise such distortions as well as would be asymmetric across the leaflets - none of which has been considered. Connecting chain length to strain energy is also not well supported - are the authors trying to correlate membrane order (Lo vs Ld) with strain energy?

(iii) Entropic effect: What is the evidence towards the entropic effect? If strain energy is entropic, the authors first need to establish that. They discuss enthalpy-entropy compensation but there is no clear data or evidence to support that argument. The lipids will rearrange themselves or have a preference to be close to certain regions of the protein and that generally arises because of enthalpies reasons (see the body of work done by Carol Robinson with Mass Spec where certain lipids prefer proteins in the GAS phase, certainly there is no entropy at play there). I find the claims of entropic effects very unconvincing.

(iv) The changes in conformations dynamics are subtle as reported by the authors and the allosteric arguments are made based on normal mode analyses. In the complex, there are large overlapping regions between the CI, CIII2, and SCI/III2. I am not sure how the allosteric crosstalk claim is established in this work - some more analyses and data would be useful. Normal mode analyses (EDA) suggest that the motions are coupled and correlated - I am not convinced that it suggests that there is allosteric cross-talk.

(v) The lattice model should be described better and the rationale for choosing the equation needs to be established. Specific interactions look unfavourable in the equation as compared to non-specific interactions.

Reviewer #3 (Public review):

Summary:

In this contribution, the authors report atomistic, coarse-grained, and lattice simulations to analyze the mechanism of supercomplex (SC) formation in mitochondria. The results highlight the importance of membrane deformation as one of the major driving forces for SC formation, which is not entirely surprising given prior work on membrane protein assembly, but certainly of major mechanistic significance for the specific systems of interest.

Strengths:

The combination of complementary approaches, including an interesting (re)analysis of cryo-EM data, is particularly powerful and might be applicable to the analysis of related systems. The calculations also revealed that SC formation has interesting impacts on the structural and dynamical (motional correlation) properties of the individual protein components, suggesting further functional relevance of SC formation. Overall, the study is rather thorough and highly creative, and the impact on the field is expected to be significant.

Weaknesses:

In general, I don't think the work contains any obvious weaknesses, although I was left with some questions.