Membrane-associated proteins (MAPs), such as channels, pumps and receptors, are notoriously difficult to study by structural methods because they require a stabilizing surrogate lipo-environment, often making sample preparation and data acquisition challenging. At the same time biological processes occurring at the cell membrane, particularly protein-protein and protein-membrane interactions, are deeply involved in homeostasis and disease; indeed, over 50% of approved pharmaceuticals target this class of cellular contacts. Thus, there is great motivation to reach a structural understanding of membrane protein biochemistry despite these objective challenges. Here we demonstrate successful applications of the lipoprotein nanodisc (LPN) technology, providing close-to-native membrane assemblies, to addressing structural questions in the membrane environment.
By incorporating the potassium channel KcsA in LPNs we could reliably identify the molecular basis of biological function in two regions of interest, the toxin-binding extracellular region and the putative pH-gating region in the cytoplasmic C-terminal domain. We used NMR to determine the structures of de novo and natural KcsA-blocking toxins, and, in combination with electrophysiology measurements, the basis for specific recognition between toxins and various channels. LPNs also enabled us to follow the tetramer-to-monomer transition in the channel’s cytoplasmic domain by NMR and EPR in terms of pH-gating, a subject of controversy in previous studies, as well as coupling to other gates. Finally, we employed LPNs to investigate host membrane-targeting by the cytotoxic effector BteA, secreted by the pathogen Bordetella pertussis responsible for causing whooping cough. Chemical shift perturbation analysis of wildtype and mutant BteA, backed by additional biophysical methods, showed that this four-helix bundle domain binds to membranes in a phosphatidylinositol-dependent manner, and defined a membrane-targeting motif that differs from that of previously described effectors. We thus demonstrate the utility of NMR methods in conjunction with LPNs in elucidating the structural biology of membrane-associated proteins.