Show Abstract ▼

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.

Open Access

Show Abstract ▼

Abstract: Despite the theoretical advantages of phosphorus single-wavelength anomalous diffraction (P-SAD) for nucleic acid phasing, its application remains limited due to high atomic displacement parameters and an unfavourable ratio of unique reflections to anomalous scatterers. In this study, we report the crystal structure of an RNA complex composed of four strands, which was solved by experimental phasing after AlphaFold3 failed to produce reliable models. Bromine single-wavelength anomalous diffraction (Br-SAD) data were collected at 0.916 Å on beamline I04 at Diamond Light Source, while phosphorus anomalous data were obtained at 3.024 Å on beamline I23. The structure was successfully phased using bromine anomalous scattering, and phosphorus anomalous peaks corroborated the backbone positions and validated the model. Attempts to phase the structure directly from phosphorus data failed, consistent with theoretical predictions that successful SAD phasing requires a significantly higher reflection-to-scatterer ratio. The final models reveal an RNA complex stabilized by Watson–Crick and Hoogsteen base pairing, forming a pseudo-helical complex instead of the anticipated hairpin stem-loop, likely reflecting crystallization artefacts. This work demonstrates the complementary use of bromine and phosphorus anomalous signals in RNA crystallography.

Show Abstract ▼

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.

Open Access

Show Abstract ▼

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.

Show Abstract ▼

Abstract: Perlecan is an essential multi-domain, disulfide bond rich basement membrane protein. Mutations in perlecan cause Schwartz-Jampel syndrome and dyssegmental dysplasia. While there has been a large body of experimental work reported on perlecan, there is only minimal structural information available to date. There is no prior structural data for region 3 of perlecan in which some Schwartz-Jampel syndrome causing point mutations have been reported. Here, we produce constructs of the disulfide rich region 3 of perlecan along with five mutations previously reported to cause Schwatz-Jampel syndrome. Four of the mutations resulted in decreased yields and thermal stability compared to the wild-type protein. In contrast, the P1019L mutation was produced in good yields and showed higher thermal stability than the wild-type protein. The crystal structures for both the wild-type and P1019L mutation were solved. As expected, both showed laminin IV-like and laminin-type EGF-like domains, with the P1019L mutation resulting in only a minor conformational change in a loop region and no significant changes in regular secondary or tertiary structure.