The alarming rise of drug-resistant bacteria urgently calls for novel antibiotics. A particularly interesting class of antibiotics target essential Lipid II molecules that are present in bacterial membranes, killing even the most refractory bacteria at nanomolecular concentrations. Unfortunately, the native binding modes of these membrane-active antibiotics are unknown due to the enormous challenge to study these drugs in a native environment. This lack of data critically limits the use of these powerful drugs for antibiotic design. Here, we present a cutting-edge solid-state NMR approach to study antibiotic-Lipid II complexes in liposomes and directly in native bacterial membranes.
Using a combination of high-field 800 MHz DNP-enhanced and 1H-detected solid-state NMR, we managed to capture the native state of the antibiotic Nisin bound to Lipid II at high resolution. Our data proof that the previous gold-standard model, a nisin – Lipid II complex acquired in artificial DMSO medium, does not represent a physiologically relevant binding mode. Furthermore, for the first time ever, we managed to capture quantitative, well-resolved 2D NMR spectra of an antibiotic directly in native bacterial cell membranes using as little as 5-10 nmol of antibiotic. These cellular experiments unravelled antibiotic domains that are responsible for adapting the Nisin-Lipid II complex to the local properties of different bacterial membranes. Intriguingly, these plastic domains also correspond to pharmaceutical hotspots of the antibiotic.
Overall our solid-state NMR approach enables the study of membrane-active drugs in their native cellular conditions with high resolution. This ultimately leads to a rational understanding of how the local bacterial membrane environment can modulate antibiotic binding and efficiency. Thereby our approach paves the way to study drug-binding and drug-resistance mechanism directly in medically relevant conditions