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 β-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.

Control of posttranscriptional mRNA decay is a crucial determinant of cell homeostasis and differentiation. mRNA lifetime is governed by cis-regulatory elements in their 3′ untranslated regions (UTR). Despite ongoing progress in the identification of cis elements we have little knowledge about the functional and structural integration of multiple elements in 3′UTR regulatory hubs and their recognition by mRNA-binding proteins (RBPs). Structural analyses are complicated by inconsistent mapping and prediction of RNA fold, by dynamics, and size. We here, for the first time, provide the secondary structure of a complete mRNA 3′UTR. We use NMR spectroscopy in a divide-and-conquer strategy complemented with SAXS, In-line probing and SHAPE-seq applied to the 3′UTR of Ox40 mRNA, which encodes a T-cell co-receptor repressed by the protein Roquin. We provide contributions of RNA elements to Roquin-binding. The protein uses its extended bi-modal ROQ domain to sequentially engage in a 2:1 stoichiometry with a 3′UTR core motif. We observe differential binding of Roquin to decay elements depending on their structural embedment. Our data underpins the importance of studying RNA regulation in a full sequence and structural context. This study serves as a paradigm for an approach in analysing structured RNA-regulatory hubs and their binding by RBPs.

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Probing the dynamics of aromatic side chains provides important insights into the behavior of a protein because flips of aromatic rings in a protein's hydrophobic core report on breathing motion involving large part of the protein. Inherently invisible to crystallography, aromatic motions have been primarily studied by solution NMR. Here we apply magic-angle spinning NMR, advanced phenylalanine 1H-13C/2H isotope labeling and molecular simulations to a protein in three different crystal packing environments to shed light onto possible impact of packing on ring flips. We find that ring flips in ubiquitin are quite conserved in the different crystal forms, even though the intermolecular packing is quite different suggesting that intramolecular influences are more important than intermolecular (packing) effects.

Aromatic side chains are important reporters of the plasticity of proteins, and often form important contacts in protein–protein interactions. We studied aromatic residues in the two structurally homologous cross‐β amyloid fibrils HET‐s, and HELLF by employing a specific isotope‐labeling approach and magic‐angle‐spinning NMR. The dynamic behavior of the aromatic residues Phe and Tyr indicates that the hydrophobic amyloid core is rigid, without any sign of “breathing motions” over hundreds of milliseconds at least. Aromatic residues exposed at the fibril surface have a rigid ring axis but undergo ring flips on a variety of time scales from nanoseconds to microseconds. Our approach provides direct insight into hydrophobic‐core motions, enabling a better evaluation of the conformational heterogeneity generated from an NMR structural ensemble of such amyloid cross‐β architecture.

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Rock‐solid amyloid?. Aromatic residues in the core of the amyloid fibrils HELLF and HET‐s show no sign of ring flips suggesting a rigid core that does not undergo "breathing motions". Aromatic residues facing outside on the other hand are highly flexible and show ring flips on various timescales. These conclusions could be drawn from dynamics measurements by solid‐state MAS NMR in combination with specific labeling of phenylalanines and tyrosines as Paul Schanda et al. describe in their Research Article (e202219314).

The rapid rise of antimicrobial resistance calls for the development of new antibiotics. Ideal templates for next-generation antibiotics could be drugs that target the cell wall precursor, Lipid II, with vancomycin, nisin, or teixobactin being well-known examples. In this chapter the authors review a selection of NMR studies on the mode of action of Lipid II-binding antibiotics with an emphasis on solid-state NMR findings in lipid membranes that are vital to obtain physiologically relevant drug-binding modes.