Juan David Orjuela

Staphyloxanthin (STX) is a carotenoid synthesized by the human pathogen Staphylococcus aureus. The golden color of this bacterium is due to this carotenoid. STX protects Staphylococcus aureus from oxidative stress by scavenging free radical species. Furthermore, STX has been shown to mechanically strengthen the Staphylococcus aureus membrane and to form microdomains that recruit antibiotic-resistance factors. Thus, inhibition of STX is a promising strategy for intervening against multidrug-resistant strains of this pathogen. However, the molecular mechanisms by which STX regulates the membrane structure and function of Staphylococcus aureus remain unclear. More specifically, the localization of STX within phosphatidylglycerol (PG) bilayers, the primary phospholipid of this bacterium's membrane, and how this localization drives macroscopic biophysical changes remain unresolved questions. Here, we addressed this issue by integrating molecular dynamics (MD) simulations, X-ray scattering experiments, and fluorescence spectroscopy. We developed an atomistic model of STX, which was validated against X-ray scattering data and which is suitable for all-atom MD simulations. We demonstrate that STX significantly increases lipid packing and acyl chain order of STX-PG bilayer mixtures. In addition, STX self-assembles into clusters, where the long and rigid conjugated triterpenoid chain interdigitates across both leaflets, modifying locally the density of the surrounding PG molecules. These findings provide a molecular explanation to the reduced headgroup spacing and core dynamics observed in fluorescence experiments and are consistent with the formation of structurally-distinct STX-enriched microdomains. Notably, STX reduces the gel-to-liquid crystalline phase transition temperature, indicating a general stabilizing effect for the fluid phase of PG lipids of varying length. Overall, our findings provide molecular insights into how STX enhances membrane mechanical integrity. It will be highly interesting to establish how the membrane remodeling effects observed here connect with STX's dual roles, acting as an antioxidant and preventing pore formation and other mechanical perturbations induced by antimicrobial molecules.

Aquaporin-0 (AQP0) tetramers form square arrays in lens membranes through a yet unknown mechanism, but lens membranes are enriched in sphingomyelin and cholesterol. Here, we determined electron crystallographic structures of AQP0 in sphingomyelin/cholesterol membranes and performed molecular dynamics (MD) simulations to establish that the observed cholesterol positions represent those seen around an isolated AQP0 tetramer and that the AQP0 tetramer largely defines the location and orientation of most of its associated cholesterol molecules. At a high concentration, cholesterol increases the hydrophobic thickness of the annular lipid shell around AQP0 tetramers, which may thus cluster to mitigate the resulting hydrophobic mismatch. Moreover, neighboring AQP0 tetramers sandwich a cholesterol deep in the center of the membrane. MD simulations show that the association of two AQP0 tetramers is necessary to maintain the deep cholesterol in its position and that 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. Since each tetramer interacts with four such ‘glue’ cholesterols, avidity effects may stabilize larger arrays. The principles proposed to drive AQP0 array formation could also underlie protein clustering in lipid rafts.

Withdrawal Statement: The authors have withdrawn this manuscript owing to its merge with BIORXIV/2023/540959. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author. The merged preprint can be found at doi.org/10.1101/2023.05.16.540959

Aquaporin-0 (AQP0) tetramers form square arrays in lens membranes through a yet unknown mechanism, but lens membranes are enriched in sphingomyelin and cholesterol. Here, we determined electron crystallographic structures of AQP0 in sphingomyelin/cholesterol membranes and performed molecular dynamics (MD) simulations to establish that the observed cholesterol positions represent those seen around an isolated AQP0 tetramer and that the AQP0 tetramer largely defines the location and orientation of most of its associated cholesterol molecules. At a high concentration, cholesterol increases the hydrophobic thickness of the annular lipid shell around AQP0 tetramers, which may thus cluster to mitigate the resulting hydrophobic mismatch. Moreover, neighboring AQP0 tetramers sandwich a cholesterol deep in the center of the membrane. MD simulations show that the association of two AQP0 tetramers is necessary to maintain the deep cholesterol in its position and that 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. Since each tetramer interacts with four such ‘glue’ cholesterols, avidity effects may stabilize larger arrays. The principles proposed to drive AQP0 array formation could also underlie protein clustering in lipid rafts.