Towards a Structural Understanding of the Mode of Action of Daptomycin: A Lipid Perspective

Abstract

Membranes form an indispensable barrier for every form of life. They contain a variety of proteins and also different lipid species. Undoubtedly, there are many interactions between lipids and between lipids and proteins. Yet, methods to study these interactions on an atomistic scale are lacking. Methodologies that allow the study of the membrane on the level of atoms will spark the discovery of drugs and antimicrobials that humanity direly needs due to rampant antimicrobial resistance. In this work we present isotopically labelled phospholipids (13C, 15N and 19F) for solid-state NMR spectroscopy (ssNMR). These probes allow the study of biologically relevant and complex membranes at atomic resolution without affecting natural membrane physics, while conventional methodologies would. We demonstrate the versatility of these probes by studying the mode of action of the lipopeptide antibiotics daptomycin and A54145, and the lantibiotic cinnamycin. We found that both these distinct antibiotic classes can form aggregates on bacterial membranes. This shows for the first time that oligomerisation as a mode of action expands beyond antibiotics that target the peptidoglycan precursor lipid II [185-187] and, thus, suggests that this is a general mode of action for membrane targeting antibiotics. Furthermore, we describe a straightforward isotopic labelling approach for daptomycin and A54145 from its producing streptomycete. Together with the isotopically labelled lipid probes, this will allow the future study of the full lipopeptide-lipid complex, which would shed light on their puzzling mode of action after more than 40 years of active research. Taken together, the lipid probes presented in this work allow for the exploration of complex membranes at an unprecedented atomic resolution, providing novel strategies for the investigation of the mode of action of drugs and antimicrobials, and others interactions within the membrane.