Cholesterol mechanically stabilizes aquaporin-0 arrays by strengthening the association of adjacent tetramers

Aquaporin 0 (AQP0) tetramers form extensive crystalline arrays in the native lens membrane that mediate the formation of membrane junctions between lens fiber cells. In the accompanying manuscript, Chiu and Walz determined the electron crystallographic structure of AQP0 arrays in sphingomyelin and cholesterol membranes, revealing several preferred cholesterol positions. Furthermore, at a high concentration, a cholesterol molecule is found near the center of the membrane, referred to as ‘deep cholesterol’, where it makes direct contacts with two adjacent AQP0 tetramers. Here, we performed molecular dynamics simulations to explore the molecular determinants for the cholesterol positions. Our simulations established that the cholesterol positions observed in the electron crystallographic structures are representative of those seen around an isolated AQP0 tetramer. Simulations also showed that an isolated AQP0 tetramer is sufficient to largely define the location and orientation of most of its associated cholesterol molecules, with little dependence on the cholesterol concentration in the membrane. However, the association of two AQP0 tetramers is necessary to maintain the deep cholesterol in its position. Remarkably, the presence of the deep cholesterol increases the force required to laterally detach two AQP0 tetramers, not only due to protein–protein contacts but also due to increased lipid–protein complementarity. Overall, our results suggest that cholesterol directly participates in the mechanical stabilization of AQP0 arrays by strengthening the lateral association of AQP0 tetramers. ### Competing Interest Statement The authors have declared no competing interest.