Engineering cardiolipin binding to an artificial membrane protein reveals determinants for lipid-mediated stabilization

Reviewer #1 (Public review):

Summary:

The study combines predictions from MD simulations with sophisticated experimental approaches including native mass spectrometry (nMS), cryo-EM, and thermal protein stability assays to investigate the molecular determinants of cardiolipin (CDL) binding and binding-induced protein stability/function of an engineered model protein (ROCKET), as well as of the native E. coli intramembrane rhomboid protease, GlpG.

Strengths:

State-of-the-art approaches and sharply focused experimental investigation lend credence to the conclusions drawn. Stable CDL binding is accommodated by a largely degenerate protein fold that combines interactions from distant basic residues with greater intercalation of the lipid within the protein structure. Surprisingly, there appears to be no direct correlation between binding affinity/occupancy and protein stability.

Weaknesses:

(i) While aromatic residues (in particular Trp) appear to be clearly involved in the CDL interaction, there is no investigation of their roles and contributions relative to the positively charged residues (R and K) investigated here. How do aromatics contribute to CDL binding and protein stability, and are they differential in nature (W vs Y vs F)? (ii) In the case of GlpG, a WR pair (W136-R137) present at the lipid-water on the periplasmic face (adjacent to helices 2/3) may function akin to the W12-R13 of ROCKET in specifically binding CDL. Investigation of this site might prove to be interesting if it indeed does. (iii) Examples of other native proteins that utilize combinatorial aromatic and electrostatic interactions to bind CDL would provide a broader perspective of the general applicability of these findings to the reader (for e.g. the adenine nucleotide translocase (ANT/AAC) of the mitochondria as well as the mechanoenzymatic GTPase Drp1 appear to bind CDL using the common "WRG' motif.)

Overall, using both model and native protein systems, this study convincingly underscores the molecular and structural requirements for CDL binding and binding-induced membrane protein stability. This work provides much-needed insight into the poorly understood nature of protein-CDL interactions.

Reviewer #2 (Public review):

Summary:

The work in this paper discusses the use of CG-MD simulations and nMS to describe cardiolipin binding sites in a synthetically designed that can be extrapolated to a naturally occurring membrane protein. While the authors acknowledge their work illuminates the challenges in engineering lipid binding they are able to describe some features that highlight residues within GlpG that may be involved in lipid regulation of protease activity, although further study of this site is required to confirm it's role in protein activity.

Comments
Discrepancy between total CDL binding in CG simulations (Fig 1d) and nMS (Fig 2b,c) should be further discussed. Limitations in nMS methodology selecting for tightest bound lipids?
Mutation of helical residues to alanine not only results in loss of lipid binding residues but may also impact overall helix flexibility, is this observed by the authors in CG-MD simulations? Change in helix overall RMSD throughout simulation? The figures shown in Fig.1H show what appear to be quite significant differences in APO protein arrangement between ROCKET and ROCKET AAXWA.
CG-MD force experiments could be corroborated experimentally with magnetic tweezer unfolding assays as has been performed for the unfolding of artificial protein TMHC2. Alternatively this work could benefit to referencing Wang et al 2019 "On the Interpretation of Force-Induced Unfolding Studies of Membrane Proteins Using Fast Simulations" to support MD vs experimental values.
Did the authors investigate if ROCKET or ROCKETAAXWA copurifies with endogenous lipids? Membrane proteins with stabilising CDL often copurify in detergent and can be detected by MS without the addition of CDL to the detergent solution. Differences in retention of endogenous lipid may also indicate differences in stability between the proteins and is worth investigation.
Do the AAXWA and ROCKET have significantly similar intensities from nMS? The AAXWA appears to show slight lower intensities than the ROCKET.
Can the authors extend their comments on why densities are observed only around site 2 in the cryo-em structures when site 1 is the apparent preferential site for ROCKET.
The authors state that nMS is consistent with CDL binding preferentially to Site 1 in ROCKET and preferentially to Site 2 in the ROCKET AAXWA variant, yet it unclear from the text exactly how these experiments demonstrate this.
As carried out for ROCKET AAXWA the total CDL binding to A61P and R66A would add to supporting information of characterisation of lipid stabilising mutations.
Did the authors investigate a double mutation to Site 2 (e.g. R66A + M16A)?
Was the stability of R66A ever compared to the WT or only to AAXWA?
How many CDL sites in the database used are structurally verified?
The work on GlpG could benefit from mutagenesis or discussion of mutagenesis to this site. The Y160F mutation has already been shown to have little impact on stability or activity (Baker and Urban Nat Chem Biol. 2012).

Reviewer #3 (Public review):

Summary:

The relationships of proteins and lipids: it's complicated. This paper illustrates how cardiolipins can stabilize membrane protein subunits - and not surprisingly, positively charged residues play an important role here. But more and stronger binding of such structural lipids does not necessarily translate to stabilization of oligomeric states, since many proteins have alternative binding sites for lipids which may be intra- rather than intermolecular. Mutations which abolish primary binding sites can cause redistribution to (weaker) secondary sites which nevertheless stabilize interactions between subunits. This may be at first sight counterintuitive but actually matches expectations from structural data and MD modelling. An analogous cardiolipin binding site between subunits is found in E.coli tetrameric GlpG, with cardiolipin (thermally) stabilizing the protein against aggregation.

Strengths:

The use of the artificial scaffold allows testing of hypothesis about the different roles of cardiolipin binding. It reveals effects which are at first sight counterintuitive and are explained by the existence of a weaker, secondary binding site which unlike the primary one allows easy lipid-mediated interaction between two subunits of the protein. Introducing different mutations either changes the balance between primary and secondary binding sites or introduced a kink in a helix - thus affecting subunit interactions which are experimentally verified by native mass spectrometry.

Weaknesses:

The artificial scaffold is not necessarily reflecting the conformational dynamics and local flexibility of real, functional membrane proteins. The example of GlpG, while also showing interesting cardiolipin dependency, illustrates the case of a binding site across helices further but does not add much to the main story. It should be evident that structural lipids can be stabilizing in more than one way depending on how they bind, leading to different and possibly opposite functional outcomes.

Author response:

(1) discuss the non-native properties of ROCKET and compare CDL binding in native proteins

ROCKET is indeed a non-native protein with exceptional stability, which makes it immune to mutations with subtle effects on structure or dynamics. We would argue that this is an advantage, allowing us to find the features with the most pronounced impact on CDL-mediated stability. The reviewers are right that there certainly are other structural features which impact CDL binding, which cannot be investigated using ROCKET. This is the reason we then apply our findings to GlpG - to translate back to native systems.

The CDL binding site geometry that we tested experimentally was derived by Corey et al (Sci Adv 2022) from large-scale computational analysis of native protein structures. Our data adds some basic rules for flexibility, which helped us to identify GlpG as a potentially CDL-regulated protein. Following the reviewers’ suggestion, we will screen the dataset from Corey et al. for experimentally confirmed examples of CDL-mediated stabilization and analyze whether they conform to the rules derived from analysis of ROCKET. In this way, we may be able to assess how general our findings are.

(2) clarify the limitations of combining MS and nMS

The reviewers correctly point out that there are differences between the MD and MS data: although the binding Site 1 has nearly 100% occupancy in MD, MS shows that ca 50% of the protein is CDL-free and that not all subunits in the tetramer have a CDL bound. Furthermore, MD shows that aromatic residues are important, but this is not tested by MS. Both points relate to the shortcomings of nMS, which requires desolvation, ionization, and detergent stripping to detect protein-lipid complexes. These processes can potentially affect lipid binding, e.g. by leading to loss of lipids that are not tightly bound. As a result, absolute quantitative comparisons between MD and MS are challenging, and contributions from subtle non-electrostatic interactions involving aromatic residues are difficult to detect. For this reason, we use relative changes in lipid interactions between different ROCKET variants to compare MD and MS data. We will discuss these factors in the revision.

(3) more detailed investigation of the structure-function relationship in GlpG-CDL complexes

We use the insights from ROCKET to identify a stabilizing CDL site in GlpG and find that CDL binding switches substrate preference from transmembrane to soluble substrates. We do not verify the binding site with mutagenesis in our study, but the MD and MS data are very unambiguous that there is only one site, and its location provides a rationale for how CDL affects substrate binding, which is described in the supplementary data.

We agree that the regulatory effect of CDL on GlpG activity raises a wide range of interesting questions relating to the mechanism of allosteric inhibition, the evolutionary background, and biological implications of E. coli using changes in membrane CDL content to steer GlpG activity. Work in our labs is on-going to investigate this further, including the mutational analysis suggested by the reviewers, but it moves beyond of the scope of the current study. We will discuss our rationale for the absence of mutagenesis data in the revision.