Several antimicrobial peptides, including magainin and the human cathelicidin LL‐37, act by forming pores in bacterial membranes. Bacteria such as Staphylococcus aureus modify their membrane's cardiolipin composition to resist such types of perturbations that compromise their membrane stability. Here, we used molecular dynamic simulations to quantify the role of cardiolipin on the formation of pores in simple bacterial‐like membrane models composed of phosphatidylglycerol and cardiolipin mixtures. Cardiolipin modified the structure and ordering of the lipid bilayer, making it less susceptible to mechanical changes. Accordingly, the free‐energy barrier for the formation of a transmembrane pore and its kinetic instability augmented by increasing the cardiolipin concentration. This is attributed to the unfavorable positioning of cardiolipin near the formed pore, due to its small polar head and bulky hydrophobic body. Overall, our study demonstrates how cardiolipin prevents membrane‐pore formation and this constitutes a plausible mechanism used by bacteria to act against stress perturbations and, thereby, gain resistance to antimicrobial agents.

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Conformational sampling is fundamentally important for simulating complex bio-molecular systems. Generalized-ensemble algorithm, especially the temperature replica-exchange molecular dynamics method (REMD), is one of the widely used methods to explore structures of bio-molecules. Most temperature REMD simulations have focused on soluble proteins rather than membrane proteins or lipid bilayers, because explicit membranes do not keep their structural integrity at high temperature. Here, we propose a new generalized-ensemble algorithm for membrane systems, which we call the surface-tension REMD method. Each replica is simulated in the NPgT ensemble, where surface tensions in a pair of replicas are exchanged at certain intervals to enhance conformational sampling of the target membrane system. We tested our method on a fully hydrated DPPC lipid bilayer. During the simulation, a random walk in surface tension space is realized. Large-scale lateral deformation of DPPC membranes takes place in all of the replicas without collapse of the lipid bilayer structure. Our method could be applicable to a wide variety of biological membrane systems including mixed lipid bilayers and membrane-protein systems.

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.