Structure and dynamics of cholesterol-mediated aquaporin-0 arrays and implications for lipid rafts

Reviewer #1 (Public Review):

Aquaporin-0 forms 2D crystals in the lens of the eye. This propensity to form 2D crystals was originally exploited to solve the structure of aquaporin-0 reconstituted in membranes. Existing structures do not explain why the proteins spontaneously form these arrays, however. In this work the authors investigate the hypothesis that the main lipids in the native membranes, sphingomyelin and cholesterol, contribute to lattice formation. By titrating the cholesterol: sphingomyelin ratio, the authors identify cholesterol binding sites of increasing stability. The authors identify a cholesterol that interacts with adjacent tetramers and is bound at an unusual membrane depth. Computational simulations suggest that this cholesterol is only stable in the context of adjacent tetramers (ie lattice formation) and that the presence of the cholesterol increases the stability of that interface. The exact mechanism is not clear, but the authors propose that the so-called "deep cholesterol" improves shape complementarity between adjacent tetramers and modulates the kinetics of protein-protein interactions. Finally, the authors provide a reasonable model for the role of cholesterol in

Strengths of this manuscript include the analysis of multiple structures determined with different lipid compositions and lipid:cholesterol ratios. For each these, multiple lipids can be modelled, giving a good sense of the lipid specificity at various favorable lipid binding positions. In addition, multiple hypotheses are tested in a very thorough computational analysis that provides the framework for interpreting the structural observations. The authors also provide a thorough scholarly discussion that connects their work with other studies of membrane protein-cholesterol interactions.

The model presented by the authors is consistent with the data described. Further testing of this model, for example by mutating the deep cholesterol binding site, would strengthen the model. However, such experiments might be challenging due to the relatively non-specific/hydrophobic nature of the deep cholesterol binding site.

Reviewer #2 (Public Review):

Summary:
In the manuscript by Chiu et al., "Structure and dynamics of cholesterol-mediated aquaporin-0 arrays and implications for lipid rafts," the authors address the effect of cholesterol on array formation by AQP0. Using a combination of electron crystallography and molecular dynamics simulations, the authors show binding of a "deep" cholesterol molecule between AQP0 tetramers. Each AQP0 tetramers binds four deep cholesterols to form a crystallographic array of AQP0.

Strengths:
The combined approaches of electron crystallography and MD simulations under different lipid conditions (different sphingomyelin and cholesterol concentrations) are a strength of the study. The authors provide a thorough and convincing assessment of cholesterol binding, protein-protein interactions, and array formation by AQP0. The MD simulations allow the authors to consider the propensity of cholesterol to occupy the observed binding sites in the absence of crystal contacts. The combined methods and the breadth of analyses set a high standard in the field of membrane protein structural biology.

The findings of the authors fit nicely into a growing body of literature on cholesterol binding sites that mediate membrane protein-protein interactions. Cholesterol interacts with a variety of membrane proteins via its smooth alpha face of rough beta face. AQP0 is somewhat unique in that it binds the rough face of cholesterol in a "deep" binding site that places cholesterol in the middle of the membrane bilayer. So-called "deep" cholesterol binding sites have been described for GPCRs and docking studies suggest they may exist on other ion channels and transporters. In the case of AQP0, the deep cholesterol acts as a glue that holds two tetramers together. Since each tetramer has four binding sites for deep cholesterol, the assembly and mechanical stability of an extended two-dimensional array of AQP0 tetramers is a natural consequence in lens membranes.

Weaknesses:
The authors report that the findings generally apply to raft formation in membranes. However, this point is less clear as the lens membrane in which AQP0 resides is rather unique in lipid and protein content and density. Nonetheless, the authors achieve the overall goal of evaluating cholesterol binding to AQP0, and there are many valuable and informative figures in the main manuscript and supplement that provide convincing results and interpretations.

Reviewer #3 (Public Review):

Summary: This manuscript aims to unravel the mechanisms behind Aquaporin-0 (AQP0) tetramer array formation within lens membranes. The authors utilized electron crystallography and molecular dynamics (MD) simulations to shed light on the role of cholesterol in shaping the structural organization of AQP0. The evidence suggests that cholesterol not only defines the positions and orientations of associated molecules but also plays a crucial role in stabilizing AQP0 tetramer arrays. This study provides valuable insights into the potential principles driving protein clustering within lipid rafts, advancing our understanding of membrane biology.

In this review, I will focus on the MD simulations part, since this is my area of expertise. The authors conducted an impressive set of MD simulations aiming at understanding the role of cholesterol in structural organization of AQP0 arrays. These simulations clearly demonstrate the well-defined localization of cholesterol molecules around a single AQP0 tetramer, aligning with previous computational studies and the crystallographic structures presented in this manuscript. Interestingly, authors identified an unusual position for one cholesterol molecule, located near the center of the lipid bilayer, which was stabilized by the adjacent AQP0 tetramers. The authors showed that these adjacent tetramers can withstand a larger lateral detachment force when deep cholesterol molecules are present at the interface compared to scenarios with sphingomyelin (SM) molecules at the interface between two AQP0 tetramers. Authors interpret that result as evidence that deep cholesterol molecules mechanically stabilize the interface of the AQP0 tetramers. This conclusion has minor weaknesses, and the rigor of the lateral detachment simulations could be increased by establishing a reference point for the detachment force needed to separate AQP0 tetramers in a scenario without lipids at the interface between tetramers, and by increasing the number of repeats for the non-equilibrium steered MD simulations. Thermodynamic integration might be a better approach to compute the stabilization energy in the presence of cholesterol compared to the SM case.