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Abstract: Voltage-gated sodium channels (VGSCs) mediate neuronal excitability and synaptic transmission and are functionally relevant targets for volatile anaesthetic (VA) actions. Here, we show that multiple VAs at clinically relevant concentrations share binding sites on NavMs, a prokaryotic VGSC. Sevoflurane, a representative VA, interacts with NavMs and NaChBac with functional effects paralleling those on human VGSCs, including modulation of channel inactivation. X-ray crystallography of purified NavMs reveals an atomic-resolution VA binding site in a VGSC, in which sevoflurane displaces lipid to occupy a membrane-embedded hydrophobic pocket. Alanine substitution of an invariant tyrosine within this binding pocket abolishes sevoflurane binding and eliminates the sevoflurane-induced hyperpolarising shift of steady-state inactivation. Sevoflurane modulates both fast and slow inactivation of human Nav1.1, demonstrating VA modulation of steady-state slow inactivation in a neuronal VGSC. Supporting evidence shows that VAs interact with homologous sites in human VGSCs. These findings define a VA binding site in VGSCs that supports a membrane-assisted pathway for modulating channel gating and neuronal activity in general anaesthesia.

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Abstract: WNK kinases are chloride- and osmotic-stress-regulated protein kinases recently shown to be controlled by potassium. Prior studies demonstrated the direct binding of chloride and osmotic stress-related water in WNK kinase regulation. Here, we probe potassium binding and regulation of WNK kinases via crystallography coupled with mutagenic analysis of WNK kinase autophosphorylation and activity. Crystals of unphosphorylated WNK1 grown in cesium formate, a surrogate for potassium, yielded nonsulfur scattering peaks at 5.75 keV. Mutations were introduced into amino acids flanking the anomalous diffraction peaks. Mutations in WNK1/E388 and the corresponding WNK3/E314, probing a peak close to WNK1/I384, led to reduced inhibition by potassium while maintaining kinase autophosphorylation and substrate phosphorylation activity. Other peaks probed by mutagenesis either did not bear out as potassium regulatory sites or were not validated due to the inactivity of the mutants synthesized. Previously synthesized chloride- and water-binding mutants demonstrate correlated sensitivity to chloride and potassium. Potassium, chloride, and water are all WNK inhibitors that share a common mechanism binding the same low-activity asymmetric dimer of WNK1 kinase domains.

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Abstract: Granulomatosis with polyangiitis is a life-threatening systemic vasculitis, characterised by anti-neutrophil cytoplasmic autoantibodies (ANCA) most commonly against proteinase 3 (PR3), a protease expressed intracellularly and on the surface of neutrophils. Most cell surface PR3 is bound to the receptor CD177; however, the molecular mechanism of the interactions is not well understood. Here, we present crystal structures of CD177 in complex with PR3 and unliganded CD177. We describe a mainly hydrophobic binding interface between PR3 and CD177, involving the first two Ly6/uPAR (LU) domains of CD177. These form a globular structure which is connected to downstream domains via a flexible linker. Using a panel of PR3-ANCA-positive patient samples, we show that a significant proportion of ANCAs target the CD177-binding site of PR3 in these samples. Structure-guided mutation of the CD177-binding site on PR3 is effective in reducing PR3-ANCA binding. The results demonstrate that the CD177-binding surface of PR3 harbours a major PR3-ANCA epitope, and that the extent of binding to this surface varies between different patients.

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Abstract: Marine picocyanobacteria are essential drivers of global biogeochemical cycles, playing a crucial role in ecosystem functioning. Their significant phylogenetic and functional diversity allow them to thrive across various marine regions. However, the mechanisms underpinning their adaptation to diverse oceanic niches remain poorly understood, especially with changing nutrient landscapes driven by climate change and anthropogenic activities. A critical factor in their success are nutrient uptake systems, particularly ATP-binding cassette (ABC) transporters, which account for over half of their transport capabilities. This thesis explores the substrate specificity, and ecological roles of substrate-binding proteins (SBPs) associated with these transporters, highlighting how these systems aid niche adaptation in marine picocyanobacteria. SBPs are key to ABC transporter function, binding substrates with high specificity and affinity, facilitating their cellular uptake. This study integrates structural biology, genomics, biophysical studies, and environmental genomics to elucidate the functions and ecological roles of these proteins, providing insights into the adaptive strategies of marine picocyanobacteria. Functional characterisation of three phosphate binding protein homologs (PstS), three urea binding protein homologs (UrtA) and two glycine-betaine binding protein homologs (ProX) from Synechococcus strains inhabiting distinct oceanic niches was conducted. The study of PstS homologs in WH8102 revealed that PstS1b, unique to Synechococcus clade III strains, is the highest affinity binding protein, providing a competitive advantage in ultraoligotrophic environments. Analysis of UrtA homologs in CC9311 and WH8102 revealed UrtA from CC9311 had higher binding affinity to urea compared to both UrtA proteins from WH8102. This is intriguing as CC9311 originates from a high-nutrient mesotrophic environment, contrasting with WH8102’s habitat in a nutrient-poor oligotrophic region. Investigation of ProX homologs from MITS9220 and WH8102 revealed a functional distinction between the two proteins, with MITS9220_ProX likely behaving as a generalist solute-binding protein, while WH8102_ProX acts as a specialist binding protein for higher salinity environments. The findings of this thesis highlight the versatility and adaptability of SBP-dependent ABC transporters in marine picocyanobacteria. The study demonstrates that these transport systems modulate their substrate affinities and specificities in response to environmental conditions, facilitating niche adaptation. This research is part of a larger project aimed at characterising the full repertoire of SBPs in marine picocyanobacteria, enhancing our understanding of nutrient acquisition mechanisms in these important microorganisms. The integration of various scientific approaches in this study provides a comprehensive framework for investigating the physiological and ecological significance of ABC uptake systems, offering valuable insights into the adaptive strategies of marine microbes in the face of changing oceanic conditions.

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Abstract: Enzymes from salt-in halophiles are stable in conditions of low water activity with applications in chiral synthesis requiring organic solvents, yet the origins of such stability remains poorly understood. Here we describe the molecular basis of the reaction mechanism and dual NADH/NADPH-specificity of D2HDH, a 2-hydroxyacid dehydrogenase from the extreme halophile Haloferax mediterranei, an organism whose proteins have to remain active in high intracellular concentrations of KCl. Halophilic adaptations of D2HDH include the expected acidic surface and a reduction in hydrophobic surface resulting from a lower lysine content. Structure determination of crystals of D2HDH grown with KCl showed that bound K+ ions were coordinated predominantly by clusters of main chain protein carbonyl ligands, with no involvement of the numerous exposed surface carboxyls. Structural comparisons identified similar sites in other halophilic proteins suggesting that the generic use of carbonyl clusters to coordinate K+ ions may also contribute in a carboxylate-independent way to the stabilisation of the folded state of the protein in its high salt environment.