Cooperative Gating of a K+ Channel by Unmodified Biological Anionic Lipids Viewed by Solid-State NMR Spectroscopy.

Lipids adhere to membrane proteins to stimulate or suppress molecular and ionic transport and signal transduction. Yet, the molecular details of lipid-protein interaction and their functional impact are poorly characterized. Here we combine NMR, coarse-grained molecular dynamics (CGMD), and functional assays to reveal classic cooperativity in the binding and subsequent activation of a bacterial inward rectifier potassium (Kir) channel by phosphatidylglycerol (PG), a common component of many membranes. Past studies of lipid activation of Kir channels focused primarily on phosphatidylinositol bisphosphate, a relatively rare signaling lipid that is tightly regulated in space and time. We use solid-state NMR to quantify the binding of unmodified 13C-PG to the K+ channel KirBac1.1 in liposomes. This specific lipid-protein interaction has a dissociation constant (Kd) of ∼7 mol percentage PG (ΧPG) with positive cooperativity (n = 3.8) and approaches saturation near 20% ΧPG. Liposomal flux assays show that K+ flux also increases with PG in a cooperative manner with an EC50 of ∼20% ΧPG, within the physiological range. Further quantitative fitting of these data reveals that PG acts as a partial (80%) agonist with fivefold K+ flux amplification. Comparisons of NMR chemical shift perturbation and CGMD simulations at different ΧPG confirm the direct interaction of PG with key residues, several of which would not be accessible to lipid headgroups in the closed state of the channel. Allosteric regulation by a common lipid is directly relevant to the activation mechanisms of several human ion channels. This study highlights the role of concentration-dependent lipid-protein interactions and tightly controlled protein allostery in the activation and regulation of ion channels.

Analyzing mechanisms of action of antimicrobial peptides on bacterial membranes requires multiple complimentary assays and different bacterial strains.

Antimicrobial peptides (AMPs) commonly target bacterial membranes and show broad-spectrum activity against microorganisms. In this research we used three AMPs (nisin, epilancin 15×, [R4L10]-teixobactin) and tested their membrane effects towards three strains (Staphylococcus simulans, Micrococcus flavus, Bacillus megaterium) in relation with their antibacterial activity. We describe fluorescence and luminescence-based assays to measure effects on membrane potential, intracellular pH, membrane permeabilization and intracellular ATP levels. The results show that our control peptide, nisin, performed mostly as expected in view of its targeted pore-forming activity, with fast killing kinetics that coincided with severe membrane permeabilization in all three strains. However, the mechanisms of action of both Epilancin 15× as well as [R4L10]-teixobactin appeared to depend strongly on the bacterium tested. In certain specific combinations of assay, peptide and bacterium, deviations from the general picture were observed. This was even the case for nisin, indicating the importance of using multiple assays and bacteria for mode of action studies to be able to draw proper conclusions on the mode of action of AMPs.

Specific Lipid Studies in Complex Membranes by Solid-State NMR Spectroscopy.

Specific interactions with phospholipids are often critical for the function of proteins or drugs, but studying these interactions at high resolution remains difficult, especially in complex membranes that mimic biological conditions. In principle, molecular interactions with phospholipids could be directly probed by solid-state NMR (ssNMR). However, due to the challenge to detect specific lipids in mixed liposomes and limited spectral sensitivity, ssNMR studies of specific lipids in complex membranes are scarce. Here, by using purified biological 13 C,15 N-labeled phospholipids, we show that we can selectively detect traces of specific lipids in complex membranes. In combination with 1 H-detected ssNMR, we show that our approach provides unprecedented high-resolution insights into the mechanisms of drugs that target specific lipids. This broadly applicable approach opens new opportunities for the molecular characterization of specific lipid interactions with proteins or drugs in complex fluid membranes.

Teixobactin kills bacteria by a two-pronged attack on the cell envelope.

Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1-3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a ß-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.