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Instruments / Technical Area	Publication Details	Published
B21-High Throughput SAXS	Elucidation of critical pH-dependent structural changes in Botulinum Neurotoxin E Christophe J. Lalaurie , Andrew Splevins , Teresa S. Barata , Karen A. Bunting , Daniel R. Higazi , Mire Zloh , Valentina A. Spiteri , Stephen J. Perkins , Paul A. Dalby Journal Of Structural Biology 12 DOI: 10.1016/j.jsb.2022.107876 Diamond Proposal Number(s): [30822] Open Access Show Abstract ▼ Abstract: Botulinum Neurotoxins (BoNT) are the most potent toxins currently known. However, they also have therapeutic applications for an increasing number of motor related conditions due to their specificity, and low diffusion into the system. Although the start- and end- points for the BoNT mechanism of action are well-studied, a critical step remains poorly understood. It is theorised that BoNTs undergo a pH-triggered conformational shift, activating the neurotoxin by priming it to form a transmembrane (TM) channel. To test this hypothesis, we combined molecular dynamic (MD) simulations and small-angle x-ray scattering (SAXS), revealing a new conformation of BoNT/E. This conformation was exclusively observed in simulations below pH 5.5, as determined by principal component analysis (PCA), and its theoretical SAXS profile matched an experimental SAXS profile obtained at pH 4. Additionally, a localised secondary structural change was observed in MD simulations below pH 5.5, in a region previously identified as instrumental for membrane insertion for BoNT/A. These changes were found at a critical pH value for BoNTs in vivo, and may be relevant for their therapeutic use.	Jun 2022
I02-Macromolecular Crystallography	The UmuC subunit of the E. coli DNA polymerase V shows a unique interaction with the â-clamp processivity factor Atif Patoli , Jody A Winter , Karen Bunting Bmc Structural Biology 13 DOI: 10.1186/1472-6807-13-12 PMID: 23822808 Open Access Show Abstract ▼ Abstract: Strict regulation of replisome components is essential to ensure the accurate transmission of the genome to the next generation. The sliding clamp processivity factors play a central role in this regulation, interacting with both DNA polymerases and multiple DNA processing and repair proteins. Clamp binding partners share a common peptide binding motif, the nature of which is essentially conserved from phage through to humans. Given the degree of conservation of these motifs, much research effort has focussed on understanding how the temporal and spatial regulation of multiple clamp binding partners is managed. The bacterial sliding clamps have come under scrutiny as potential targets for rational drug design and comprehensive understanding of the structural basis of their interactions is crucial for success.	Jun 2013
I03-Macromolecular Crystallography	The crystal structure of Haloferax volcanii proliferating cell nuclear antigen reveals unique surface charge characteristics due to halophilic adaptation Jody A. Winter , Panayiotis Christofi , Shaun Morroll , Karen A. Bunting Bmc Structural Biology 9 (55) DOI: 10.1186/1472-6807-9-55 PMID: 19698123 Open Access Show Abstract ▼ Abstract: Background The high intracellular salt concentration required to maintain a halophilic lifestyle poses challenges to haloarchaeal proteins that must stay soluble, stable and functional in this extreme environment. Proliferating cell nuclear antigen (PCNA) is a fundamental protein involved in maintaining genome integrity, with roles in both DNA replication and repair. To investigate the halophilic adaptation of such a key protein we have crystallised and solved the structure of Haloferax volcanii PCNA (Hv PCNA) to a resolution of 2.0 Å. Results The overall architecture of Hv PCNA is very similar to other known PCNAs, which are highly structurally conserved. Three commonly observed adaptations in halophilic proteins are higher surface acidity, bound ions and increased numbers of intermolecular ion pairs (in oligomeric proteins). Hv PCNA possesses the former two adaptations but not the latter, despite functioning as a homotrimer. Strikingly, the positive surface charge considered key to PCNA's role as a sliding clamp is dramatically reduced in the halophilic protein. Instead, bound cations within the solvation shell of Hv PCNA may permit sliding along negatively charged DNA by reducing electrostatic repulsion effects. Conclusion The extent to which individual proteins adapt to halophilic conditions varies, presumably due to their diverse characteristics and roles within the cell. The number of ion pairs observed in the Hv PCNA monomer-monomer interface was unexpectedly low. This may reflect the fact that the trimer is intrinsically stable over a wide range of salt concentrations and therefore additional modifications for trimer maintenance in high salt conditions are not required. Halophilic proteins frequently bind anions and cations and in Hv PCNA cation binding may compensate for the remarkable reduction in positive charge in the pore region, to facilitate functional interactions with DNA. In this way, Hv PCNA may harness its environment as opposed to simply surviving in extreme halophilic conditions.	Aug 2009

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