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Abstract: The enterobacterial common antigen (ECA) is conserved in Gram-negative bacteria of the Enterobacterales order although its function is debated. ECA biogenesis depends on the Wzx/Wzy-dependent strategy whereby the newly synthesized lipid-linked repeat units, lipid III, are transferred across the inner membrane by the lipid III flippase WzxE. WzxE is part of the Wzx family and required in many glycan assembly systems, but an understanding of its molecular mechanism is hindered due to a lack of structural evidence. Here, we present the first X-ray structures of WzxE from Escherichia coli in complex with nanobodies. Both inward- and outward-facing conformations highlight two pairs of arginine residues that move in a reciprocal fashion, enabling flipping. One of the arginine pairs coordinated to a glutamate residue is essential for activity along with the C-terminal arginine rich tail located close to the entrance of the lumen. This work helps understand the translocation mechanism of the Wzx flippase family.

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Abstract: One of the challenges for experimental structural biology in the 21st century is to see chemical reactions happen. Staphylococcus aureus (S. aureus) DNA gyrase is a type IIA topoisomerase that can create temporary double-stranded DNA breaks to regulate DNA topology. Drugs, such as gepotidacin, zoliflodacin and the quinolone moxifloxacin, can stabilize these normally transient DNA strand breaks and kill bacteria. Crystal structures of uncleaved DNA with a gepotidacin precursor (2.1 Å GSK2999423) or with doubly cleaved DNA and zoliflodacin (or with its progenitor QPT-1) have been solved in the same P61 space-group (a = b ≈ 93 Å, c ≈ 412 Å). This suggests that it may be possible to observe the two DNA cleavage steps (and two DNA-religation steps) in this P61 space-group. Here, a 2.58 Å anomalous manganese dataset in this crystal form is solved, and four previous crystal structures (1.98 Å, 2.1 Å, 2.5 Å and 2.65 Å) in this crystal form are re-refined to clarify crystal contacts. The structures clearly suggest a single moving metal mechanism—presented in an accompanying (second) paper. A previously published 2.98 Å structure of a yeast topoisomerase II, which has static disorder around a crystallographic twofold axis, was published as containing two metals at one active site. Re-refined coordinates of this 2.98 Å yeast structure are consistent with other type IIA topoisomerase structures in only having one metal ion at each of the two different active sites.

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Abstract: Metal ions have important roles in supporting the catalytic activity of DNA-regulating enzymes such as topoisomerases (topos). Bacterial type II topos, gyrases and topo IV, are primary drug targets for fluoroquinolones, a class of clinically relevant antibacterials requiring metal ions for efficient drug binding. While the presence of metal ions in topos has been elucidated in biochemical studies, accurate location and assignment of metal ions in structural studies have historically posed significant challenges. Recent advances in X-ray crystallography address these limitations by extending the experimental capabilities into the long-wavelength range, exploiting the anomalous contrast from light elements of biological relevance. This breakthrough enables us to confirm experimentally the locations of Mg2+ in the fluoroquinolone-stabilized Streptococcus pneumoniae topo IV complex. Moreover, we can unambiguously identify the presence of K+ and Cl- ions in the complex with one pair of K+ ions functioning as an additional intersubunit bridge. Overall, our data extend current knowledge on the functional and structural roles of metal ions in type II topos.

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Abstract: The insulin-linked polymorphic region is a variable number of tandem repeats region of DNA in the promoter of the insulin gene that regulates transcription of insulin. This region is known to form the alternative DNA structures, i-motifs and G-quadruplexes. Individuals have different sequence variants of tandem repeats and although previous work investigated the effects of some variants on G-quadruplex formation, there is not a clear picture of the relationship between the sequence diversity, the DNA structures formed, and the functional effects on insulin gene expression. Here we show that different sequence variants of the insulin linked polymorphic region form different DNA structures in vitro. Additionally, reporter genes in cellulo indicate that insulin expression may change depending on which DNA structures form. We report the crystal structure and dynamics of an intramolecular i-motif, which reveal sequences within the loop regions forming additional stabilising interactions that are critical to formation of stable i-motif structures. The outcomes of this work reveal the detail in formation of stable i-motif DNA structures, with potential for rational based drug design for compounds to target i-motif DNA.

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Abstract: Bacteria form sessile antimicrobial-tolerant communities called biofilms, threatening health, infrastructure, and the environment. Pseudomonas aeruginosa is a biofilm-forming opportunistic pathogen, responsible for causing many chronic infections, particularly in the lungs of Cystic Fibrosis patients. Environmental stimuli regulate biofilm dispersal to benefit the survival of the bacterial cells. Redox changes regulate biofilm dispersal, and nitric oxide has a role in this process. Several proteins respond to redox changes to disperse the biofilm, and these often contain the sensory PAS domains. The complete pathway of this redox-stimulated biofilm dispersal is currently not fully comprehended. Within this thesis the concept of bacterial biofilms and the involvement of redox in their lifecycle is explored. With a focus on PAS domains, several proteins which are redox-responding and regulate biofilm lifecycle are studied, namely BdlA, PipA, PA2072, and RbdA. Firstly, analysis was completed to further a bioinformatical study to predict the functions of lesser researched proteins in P. aeruginosa. Comparing the PAS domains phylogenetically and sequentially to a reference set of structurally characterised PAS domains generated testable hypotheses and highlighted PAS domains for which the ligand/cofactor has not been yet discovered in the P. aeruginosa genome; some of which were followed up with structure prediction analysis. BdlA, a biofilm dispersal protein with two PAS domains, was identified to have a disulphide bond which could be responsible for the reaction to redox changes. This disulphide was mutated, and the structure solved, indicating movement of residues surrounding this bond. PipA, a phage-inducing phosphodiesterase which can disperse the biofilm, has two PAS domains, the structures of which were each individually solved. The PAS1 domain is shown not to have a cofactor/ligand bound but to have a large cavity of interest. The PAS2 domain non-covalently binds an FAD cofactor, shown via UV-visible spectroscopy to be able to chemically reduce in solution and photoreduce as a cause of X-rays in crystallo, but the structure of the flavin did not vastly differ between the oxidised and reduced states seen at synchrotrons or XFEL sources. However, there was some evidence of minor change occurring in X-ray pump-probe analysis which. Finally, PA2072 and RbdA are compared and contrasted for their similar domain composition and opposite functionalities, including comparison of PAS and periplasmic domains with structural prediction highlighting key differences and a potential mechanism is unveiled. The structure of the EAL domain of the RbdA protein in a catalytically primed dimer form is shown. Overall, this thesis contributes to further understanding of PAS domains, and adds further insight into how redox induced bacterial biofilm dispersal is conducted.