Analysis of Protein-Cardiolipin Interactions in the E. coli Inner Membrane

Abstract

Integral membrane proteins are localised and functionally regulated by lipids present in the surrounding bilayer. Whilst bacteria such as E. coli have a relatively simple membrane when compared to eukaryotic cells, there is ample evidence that multiple proteins bind, and have their function regulated by, specific lipid interactions. There are a number of ways of identifying protein-lipid interactions in membrane proteins, including through molecular simulation. Here, we highlight the capabilities of this technique by running lipid-binding simulations of 42 different E. coli inner membrane proteins. Our data reveals a strong cross-membrane asymmetry in the nature of protein-lipid interactions, with a marked increase of anionic lipid binding to the inner leaflet regions of membrane proteins. This appears to be driven by an increase in basic residues, suggesting a structural basis for the positive inside rule. We have identified ca. 700 independent cardiolipin binding sites from our data, allowing us dissect the molecular basis of a prototypical cardiolipin headgroup binding site. Applying free energy calculations quantifies the relative contributions of each residue type to cardiolipin binding. These findings demonstrate the potential for large-scale simulations to reveal pertinent underlying biological information applying to a number of systems, and paves the way for future analyses of more complex protein-lipid interactions.