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Abstract: Pathogens have evolved diverse strategies to counteract host immunity. Ubiquitylation of lipopolysaccharide (LPS) on cytosol-invading bacteria by the E3 ligase RNF213 creates ‘eat me’ signals for antibacterial autophagy, but whether and how cytosol-adapted bacteria avoid LPS ubiquitylation remains poorly understood. Here, we show that the enterobacterium Shigella flexneri actively antagonizes LPS ubiquitylation through IpaH1.4, a secreted effector protein with ubiquitin E3 ligase activity. IpaH1.4 binds to RNF213, ubiquitylates it and targets it for proteasomal degradation, thus counteracting host-protective LPS ubiquitylation. To understand how IpaH1.4 recognizes RNF213, we determined the cryogenic electron microscopy structure of the IpaH1.4–RNF213 complex. The specificity of the interaction is achieved through the leucine-rich repeat of IpaH1.4, which binds the RING domain of RNF213 by hijacking the conserved RING interface required for binding to ubiquitin-charged E2 enzymes. IpaH1.4 also targets other E3 ligases involved in inflammation and immunity through binding to the E2-interacting face of their RING domains, including the E3 ligase LUBAC that is required for the synthesis of M1-linked ubiquitin chains on cytosol-invading bacteria downstream of RNF213. We conclude that IpaH1.4 has evolved to antagonize multiple antibacterial and proinflammatory host E3 ligases.

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Abstract: Auxins are a group of phytohormones that control plant growth and development. Their crucial role in plant physiology has inspired development of potent synthetic auxins that can be used as herbicides. Phenoxyacetic acid derivatives are a widely used group of auxin herbicides in agriculture and research. Despite their prevalence, the identity of the transporters required for distribution of these herbicides in plants is both poorly understood and the subject of controversial debate. Here we show that PIN-FORMED auxin transporters transport a range of phenoxyacetic acid herbicides across the membrane. We go on to characterize the molecular determinants of substrate specificity using a variety of different substrates as well as protein mutagenesis to probe the binding site. Finally, we present cryogenic electron microscopy structures of Arabidopsis thaliana PIN8 bound to either 2,4-dichlorophenoxyacetic acid or 4-chlorophenoxyacetic acid. These structures represent five key states from the transport cycle, allowing us to describe conformational changes associated with the transport cycle. Overall, our results reveal that phenoxyacetic acid herbicides use the same export machinery as endogenous auxins and exemplify how transporter binding sites undergo transformations that dictate substrate specificity. These results provide a foundation for future development of novel synthetic auxins and for precision breeding of herbicide-resistant crop plants.

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Abstract: Uncoupling protein 1 (UCP1) in the mitochondrial inner membrane is responsible for the thermogenic properties of brown adipose tissue. In response to cold exposure, UCP1 is activated by free fatty acids and catalyses proton leak, dissipating the proton motive force, which mitochondria use to drive ATP synthesis. Therefore, UCP1 uncouples the oxidation of nutrients from the phosphorylation of ADP, releasing heat instead of generating ATP. The thermogenic properties of UCP1 are regulated by cytosolic purine nucleotides, which inhibit proton conductance and therefore enable activation only in response to cold environments. This thesis aimed to determine the first high-resolution structure of UCP1 to address several unanswered questions about its overall structure and its nucleotide inhibition and activation mechanisms. Initially, attempts were made to grow three-dimensional crystals for X-ray crystallography, but later cryogenic electron microscopy (cryo-EM) and single particle analysis were used to determine the structure. Human UCP1 was purified from yeast mitochondria and modified with nanobody fiducials to develop a sample suitable for cryo-EM analysis. The resolved structure revealed that UCP1 has a similar fold to the mitochondrial ADP/ATP carrier in the cytoplasmic-open state, consisting of six transmembrane and three small matrix helices, and three bound cardiolipin molecules. The purine nucleotide binds in the central cavity, which is open to the intermembrane space, through ionic, polar and cation-π interactions with the positively charged central cavity. The structure clarifies the structural basis of the pH-dependency of nucleotide binding. The negatively charged phosphate groups of the nucleotide are positioned close to the negatively charged residues of the matrix gate, requiring proton-mediated bonds to prevent the electrostatic repulsion that occurs at higher pH levels. UCP1 has retained all of the key functional and structural features required for a mitochondrial carrier–like transport mechanism. Structural analyses show that inhibitor binding prevents the conformational changes that UCP1 might use to facilitate proton leak. Further analysis revealed that the nucleotide-binding specificity of UCP1 is not limited to purine nucleotides, but also includes pyrimidine nucleotides. Thus, all cytosolic nucleotides inhibit UCP1 proton conductance, regardless of the nucleobase, with only subtle differences in binding affinities.

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Abstract: The molecular details of phage tail contraction and bacterial cell envelope penetration remain poorly understood and are completely unknown for phages infecting bacteria enveloped by proteinaceous S-layers. Here, we reveal the extended and contracted atomic structures of an intact contractile-tailed phage (φCD508) that binds to and penetrates the protective S-layer of the Gram-positive human pathogen Clostridioides difficile. The tail is unusually long (225 nm), and it is also notable that the tail contracts less than those studied in related contractile injection systems such as the model phage T4 (∼20% compared with ∼50%). Surprisingly, we find no evidence of auxiliary enzymatic domains that other phages exploit in cell wall penetration, suggesting that sufficient energy is released upon tail contraction to penetrate the S-layer and the thick cell wall without enzymatic activity. Instead, the unusually long tail length, which becomes more flexible upon contraction, likely contributes toward the required free energy release for envelope penetration.

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Abstract: GPR55 is an orphan G protein-coupled receptor (GPCR) and represents a promising drug target for cancer, inflammation, and metabolic diseases. The endogenous activation of lipid GPCRs can be solely mediated by membrane components and different lipids have been proposed as endogenous activators of GPR55, such as cannabinoids and lysophosphatidylinositols. Here, we determine high-resolution cryo-electron microscopy structures of the activated GPR55 in complex with heterotrimeric G13 and two structurally diverse ligands: the putative endogenous agonist 1-palmitoyl-2-lysophosphatidylinositol (LPI) and the synthetic agonist ML184. These results reveal insights into ligand recognition at GPR55, G protein coupling and receptor activation. Notably, an orthosteric binding site opening towards the membrane is observed in both structures, enabling direct interaction of the agonists with membrane lipids. The structural observations are supported by mutagenesis and functional experiments employing G protein dissociation assays. These findings will be of importance for the structure-based development of drugs targeting GPR55.