Extensive substrate recognition by the streptococcal antibody-degrading enzymes IdeS and EndoS

Abigail Sudol – University of Southampton

Enzymatic cleavage of IgG antibodies is a common strategy used by pathogenic bacteria to ablate immune effector function. The Streptococcus pyogenes bacterium secretes the protease IdeS and the glycosidase EndoS, which specifically catalyse cleavage and deglycosylation of human IgG, respectively. IdeS has received clinical approval for kidney transplantation in hypersensitised individuals, while EndoS has found application in engineering antibody glycosylation. We present crystal structures of both enzymes in complex with their IgG1 Fc substrate, which was achieved using Fc engineering to disfavour preferential Fc crystallisation. The IdeS protease displays extensive Fc recognition and encases the antibody hinge. Conversely, the glycan hydrolase domain in EndoS traps the Fc glycan in a “flipped-out” conformation, while additional recognition of the Fc peptide is driven by the so-called carbohydrate binding module. In this work, we reveal the molecular basis of antibody recognition by bacterial enzymes, providing a template for the development of next-generation enzymes.

Using ColabFold and Molecular Dynamics to Elucidate Insecticidal Protein Structure and Mechanism

Adam Cutts – Cardiff University

Introduction: Bacillus thuringiensis (Bt) is a bacterium that produces a broad range of insecticidal proteins, many of which have resolved structures, including Tpp80Aa1,(1) Cry11Aa1,(2) and Mpp75Aa1.(3) Of particular interest are the vegetative insecticidal proteins (Vips) which exhibit insecticidal properties against Lepidoptera. Members of Lepidoptera, particularly from the genus Spodoptera, are agricultural pests requiring decisive measures to manage the significant economic risks they pose.(4,5) Currently, Vip3Aa16 and Vip3Bc1 have protoxin and activated structures available.(6,7) However, the activated forms lack resolved structures for the extended Domain 1 assembly. Additionally, the exact mechanism through which Vips exert their toxic effect is poorly understood. This project aims to elucidate these mechanisms.

Methods: ColabFold was employed to produce predicted structures of the extended Domain 1 assembly, which were integrated into the existing crystal/Cryo-EM structures to provide complete Vip models. GROMACS was employed to conduct molecular dynamics simulations to explore the interactions of Vips with membranes and how Vips lead to cell/insect death. Membrane:protein systems were generated using the CHARMM-GUI interface.

Results: Vips demonstrate an extended (~200 nm) coiled-coil composed of 4 ⍺-helices, with Vip3A variants exhibiting an unstructured region near the N-terminus which could convey flexibility or promote rotational movements of the terminal residues. The putative transmembrane region displays a hydrophilic exterior and hydrophobic interior. Vip3Bc1 also displays an unstructured region. Molecular dynamics simulations demonstrate the ability of water to move through the extended Domain 1 assembly, suggesting cell death is mediated via pore formation and cell leakage.

Conclusion: Evidence suggests Vips exert toxicity via pore formation mediated by a 200 nm needle-like structure which embeds into target membranes.

1.     Best HL, Williamson LJ, Lipka-Lloyd M, Waller-Evans H, Lloyd-Evans E, Rizkallah PJ, et al. The Crystal Structure of Bacillus thuringiensis Tpp80Aa1 and Its Interaction with Galactose-Containing Glycolipids. Toxins (Basel). 2022 Dec 1;14(12):863.

2.     Tetreau G, Sawaya MR, De Zitter E, Andreeva EA, Banneville AS, Schibrowsky NA, et al. De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals. Nature Communications 2022 13:1. 2022 Jul 28;13(1):1–18.

3.     Kouadio JL, Duff S, Aikins M, Zheng M, Rydel T, Chen D, et al. Structural and functional characterization of Mpp75Aa1.1, a putative beta-pore forming protein from Brevibacillus laterosporus active against the western corn rootworm. PLoS One. 2021 Oct 1;16(10):e0258052.

4.     Lázaro-Berenguer M, Quan Y, Hernández-Martínez P, Ferré J. In vivo competition assays between Vip3 proteins confirm the occurrence of shared binding sites in Spodoptera littoralis. Nature Scientific Reports. 2022;12(4578).

5.     Goergen G, Kumar PL, Sankung SB, Togola A, Tamò M. First Report of Outbreaks of the Fall Armyworm Spodoptera frugiperda (Lepidoptera, Noctuidae), a New Alien Invasive Pest in West and Central Africa. PLoS One. 2016;11(10).

6.     Núñez-Ramírez R, Huesa J, Bel Y, Ferré J, Casino P, Arias-Palomo E. Molecular architecture and activation of the insecticidal protein Vip3Aa from Bacillus thuringiensis. Nat Commun. 2020 Dec 1;11(1).

7.     Byrne MJ, Iadanza MG, Perez MA, Maskell DP, George RM, Hesketh EL, et al. Cryo-EM structures of an insecticidal Bt toxin reveal its mechanism of action on the membrane. Nat Commun. 2021 Dec 1;12(1:2791).

Nanocrystallography – collecting data from 1-micron crystals at VMXm

Anna Warren – Diamond Light Source

The Versatile Macromolecular Crystallography Microfocus (VMXm) beamline is a new micro/nanofocus beamline joining the suite of macromolecular crystallography beamlines at Diamond Light Source.  The beamline has been designed to enable rotation data collection from microcrystals down to 0.5 microns in size and took its first users in Autumn 2018.  The beamline optics deliver a beamsize of 0.3 – 10 μm vertically and horizontally between 0.5 – 5 μm to the sample position. The beamline operates at energies between 6-28 keV, delivering ~1012 ph/s to the sample (at 12.5 keV). The beamline is equipped with two detectors, a Pilatus3 6M (Si sensor) and Eiger2 X 9M (CdTe sensor) which are fully interchangeable. With the CdTe detector the quantum efficiency of the detector is improved at higher energies compared to the Si detector. With this in mind, photoelectron escape can be exploited in microcrystals using higher energy data collections, and this in turn can prolong the lifetime of the crystals in the beam.
Microcrystals are prepared on electron microscopy grids using techniques borrowed from cryo-EM.  Samples are prepared in advance using the VMXm support laboratory, with grid preparation being validated through image visualization on an offline Scanning Electron Microscope (SEM). Once grids are introduced to the beamline, crystals are visualized and aligned to the X-ray beam using either an on-axis optical microscope or a built-in SEM.  Signal to noise of the diffracted X-rays is greatly improved due to the mounting technique of the crystals, and the samples being held under vacuum for diffraction measurements, reducing background scatter to a minimum.
An outline of the beamline will be shown along with recent data collection results. The beamline is currently in a commissioning with users’ phase, successfully collecting data on crystals ranging from 2 µm up in size. The first novel structure has been solved using Se derivatized samples. We have also seen improvements in microcrystal data collections exploiting photoelectron escape and prolonging the lifetime of crystals.

Programmed iteration controls the assembly of the nonanoic acid side chain of the antibiotic mupirocin

Ash Winter – University of Bristol

Ashley J. Winter,[a] Matthew T. Rowe, [a]  Angus N. M. Weir,[a]  Nahida Akter,[a]  Sbusisiwe Z. Mbatha,[a]  Paul D. Walker, [a]  Christopher Williams,[a]  Zhongshu Song,[a]  Paul R. Race,[b] and Christine L. Willis,[a] Matthew P. Crump[a]
[a] School of Chemistry, University of Bristol, Bristol (UK), [b] School of Biochemistry, University of Bristol, Bristol (UK)
Mupirocin is a mixture of polyketides known as pseudomonic acids (PA) A to C, where PA-A is the major component. It is produced by Pseudomonas fluorescens NCIMB 10586 and this clinically important antibiotic has specific activity against methicillin resistant Staphylococcus aureus (MRSA). Mupirocin is assembled by a complex trans-acyltransferase polyketide synthase (PKS), with the biosynthetic gene cluster containing six large open reading frames (ORFs) encoding  type I PKS modules (mmpA to mmpF), 30 ORFs encoding mupA-Z and five trans-acting acyl carrier proteins (ACPs): macpA-E.1
The molecular framework of mupirocin consists of two fragments: a C17 monic acid polyketide containing a tetrahydropyran core and a 9-hydroxynonanoic acid (9HN) side chain, which is esterified to the polyketide fragment. 9HN is essential for biological activity and its unusual assembly is initialised from a 3-hydroxyproprionate starter unit bound to a dedicated ACP (3HP-MacpD). The mechanism of 9HN formation from 3HP-MacpD unit is unknown.2
We have studied the fate of the 3HP-MacpD species via the application of NMR spectroscopy, mass spectrometry, chemical probes and in vitro assays to establish the remaining steps of 9HN biosynthesis.3 This investigation revealed a complex interplay between a novel iterative or “stuttering“ KS-AT didomain (MmpF), the multidomain type 1 module MmpB and multiple ACPs. Our analysis revealed important implications for understanding the late-stage biosynthetic steps of mupirocin, future engineering of related trans-AT biosynthetic pathways (e.g. thiomarinol) and underpinning the mechanistic steps of esterification.

References
1. A. K. El-Sayed, J. Hothersall, S. M. Cooper, E. Stephens, T. J. Simpson and C. M. Thomas, Characterization of the mupirocin biosynthesis gene cluster from Pseudomonas fluorescens NCIMB 10586, Chem Biol, 2003, 10, 419-430.
2. P. D. Walker, M. T. Rowe, A. J. Winter, A. N. M. Weir, N. Akter, L. Wang, P. R. Race, C. Williams, Z. Song, T. J. Simpson, C. L. Willis and M. P. Crump, A Priming Cassette Generates Hydroxylated Acyl Starter Units in Mupirocin and Thiomarinol Biosynthesis, ACS Chemical Biology, 2020, 15, 494-503.
3. A. J. Winter, M. T. Rowe, A. N. M. Weir, N. Akter, S. Z. Mbatha, P. D. Walker, C. Williams, Z. Song, P. R. Race, C. L. Willis and M. P. Crump, Programmed Iteration Controls the Assembly of the Nonanoic Acid Side Chain of the Antibiotic Mupirocin, Angewandte Chemie International Edition, 2022, 61, e202212393.

Structural studies in support of the development of the new antibiotic gepotidacin

Ben Bax – Medicines Discovery Institute, Cardiff University

S. aureus DNA gyrase is a type IIA topoisomerase that creates a temporary four base-pair staggered double-stranded DNA break to regulate DNA topology. Quinolone drugs such as moxifloxacin (1-3), and spirocyclicpyridmidinetriones (SPTs) such as zoliflodacin (4), stabilize these normally transient double-strand DNA-strand breaks by binding at the two (four base-pair separated) DNA-cleavage sites sterically blocking DNA-religation. Here we discuss the generic hERG liability (5) of the NBTI class (6) and how this has been overcome (7) in the development of the new antibiotic gepotidacin (8, 9). Potential structurally based avenues for overcoming the generic hERG liabilities of the NBTI class will be proposed, so that other safe and efficacious new antibiotics can be developed.
1. Wohlkonig A, Chan PF, Fosberry AP, Homes P, Huang J, Kranz M, et al. Structural basis of quinolone inhibition of type IIA topoisomerases and target-mediated resistance. Nat Struct Mol Biol. 2010;17(9):1152-3.
2. Chan PF, Srikannathasan V, Huang J, Cui H, Fosberry AP, Gu M, et al. Structural basis of DNA gyrase inhibition by antibacterial QPT-1, anticancer drug etoposide and moxifloxacin. Nat Commun. 2015;6:10048.
3. Bax BD, Murshudov G, Maxwell A, Germe T. DNA Topoisomerase Inhibitors: Trapping a DNA-Cleaving Machine in Motion. J Mol Biol. 2019;431(18):3427-49.
4. Morgan H, Lipka-Lloyd M, Warren AJ, Hughes N, Holmes J, Burton NP, et al. A 2.8 Å Structure of Zoliflodacin in a DNA Cleavage Complex with Staphylococcus aureus DNA Gyrase. International Journal of Molecular Sciences. 2023;24(2):1634.
5. Kolaric A, Minovski N. Novel bacterial topoisomerase inhibitors: challenges and perspectives in reducing hERG toxicity. Future Med Chem. 2018;10(19):2241-4.
6. Bax BD, Chan PF, Eggleston DS, Fosberry A, Gentry DR, Gorrec F, et al. Type IIA topoisomerase inhibition by a new class of antibacterial agents. Nature. 2010;466(7309):935-40.
7. Hossain M, Zhou M, Tiffany C, Dumont E, Darpo B. A Phase I, Randomized, Double-Blinded, Placebo- and Moxifloxacin-Controlled, Four-Period Crossover Study To Evaluate the Effect of Gepotidacin on Cardiac Conduction as Assessed by 12-Lead Electrocardiogram in Healthy Volunteers. Antimicrob Agents Chemother. 2017;61(5).
8. Gibson EG, Bax B, Chan PF, Osheroff N. Mechanistic and Structural Basis for the Actions of the Antibacterial Gepotidacin against Staphylococcus aureus Gyrase. ACS Infect Dis. 2019;5(4):570-81.
9. Arends SR, Butler D, Scangarella-Oman N, Castanheira M, Mendes RE. Antimicrobial Activity of Gepotidacin Tested against Escherichia coli and Staphylococcus saprophyticus Isolates Causing Urinary Tract Infections in Medical Centers Worldwide (2019 to 2020). Antimicrobial Agents and Chemotherapy. 2023;67(4):e01525-22.

Understanding redox induced biofilm dispersal

Charlotte Cordery – University of Southampton/Diamond Light Source

Biofilms are communities of bacterial cells which are surrounded by an extracellular polymeric substance attached to a surface or each other. They are antibiotic tolerant and cause huge threats to health, infrastructure and the environment. Biofilms form when c-di-GMP increases in concentration and disperse when c-di-GMP concentration is decreased, regulated by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) respectively. These are often found as tandem domains often accompanied by a sensory domain such as a PAS domain. Due to the heterogeneous nature of the biofilm we sought to understand how redox stimuli can cause biofilm dispersal or formation using structural biology.
We have studied 4 different proteins to understand more about the biofilm lifecycle switching mechanisms, particularly in response to redox stimuli. These proteins all contain PAS domains and some have periplasmic sensory domains. We made predictions on the functions of PAS domains based on their predicted binding cofactor/ligand from the phylogenetic and structural comparison analysis. We can begin to make some assumptions into the mechanisms of some of these proteins from the studies we have completed.

Investigating the formation of biologically relevant DNA i-motifs

Daniela Lopes Cardoso – University of Portsmouth

Daniela Lopes Cardoso(1), Fiona Meyers(2), Garry Scarlett(2), John Brazier(3), Matt Guille(2) and Samuel Robson(1)
(1) School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth UK
(2) School of Biological Sciences, University of Portsmouth, Portsmouth UK
(3) School of Pharmacy, University of Reading, Reading UK

The i-motif is a non-standard DNA structure, very distinct from the conventional B-form double-helix shape. It was previously believed that the i-motif structure was not biologically relevant due to its requirement for an acidic environment, typically not seen inside cells. However, recent findings provide evidence that some DNA sequences form i-motif structures at intra-cellular pH conditions and that these can be directly observed within genomes of living cells. For instance, i-motifs found within gene promoters are involved in the regulation of gene expression, including transcriptional up-regulation by providing a platform for transcription factor binding, and down-regulation by disrupting nucleosome formation.
Whilst a sequence-specific prediction of i-motifs has been proposed, this approach certainly overestimates the abundance of i-motifs present in cells, while missing potential i-motif forming sequences that deviate from this predicted pattern. To fully understand the functional significance of i-motifs, it is imperative to validate and establish a complete map of their actual distribution throughout the genome and the conditions under which they exist within living organisms. In this project, we aim to provide a genome-wide map of i-motifs in the vertebrate genome of Xenopus tropicalis and explore their transitory nature throughout development. This will provide essential knowledge towards improving predictive algorithms, as well as providing evidence of their functional role.
Initially, we have combined computational prediction and in vitro experimental analysis to validate and characterize a number of predicted i-motif sequences obtained from the Xenopus tropicalis genome. These putative i-motif sequences were screened against curated i-motif sequences by utilizing biochemical and biophysical techniques, such as specific antibody binding for validation and circular dichroism (CD) spectroscopy for further structural characterization. We will then identify i-motif sequences genome-wide in vivo by making use of an antibody that specifically recognises i-motifs over other DNA structures, employing a customised CUT&RUN approach to Xenopus tropicalis cell lines and embryos in different developmental stages. This research project will generate the first experimentally validated in vivo map of i-motifs in the vertebrate genome.

Structural and biological insights into novel agents for PET imaging and targeted drug delivery to αvβ6-positive tumours

Emma Swift – Viral Immunotherapies and Advanced Therapeutics Lab, Cardiff University

Emma A. Swift (1), Stephen J. Paisey (2), Toby J. Phesse (3),  John F. Marshall (4), Pierre J. Rizkallah (5), Alan L. Parker (1), Rebecca Bayliss (1)
1. Division of Cancer and Genetics, School of Medicine, Cardiff University, Cardiff, CF14 4XN, UK
2. Wales Research and Diagnostic Positron Emission Tomography Imaging Centre, School of Medicine, Cardiff University, Cardiff, CF14 4XN, UK 
3. European Cancer Stem Cell Research Institute, Cardiff University, Cardiff, CF24 4HQ, UK
4. Centre for Tumour Biology, Barts Cancer Institute, Queen Mary University of London, London, EC1M 6BQ, UK
5. Division of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, CF14 4XN, UK

αvβ6 integrins are minimally expressed on the healthy human epithelium, but upregulated in several aggressive epithelial carcinomas, marking them out as a favourable target for development of diagnostic agents and anti-cancer therapeutics (1). Several classes of biologic capable of selectively recognising this integrin are currently being investigated, including short peptide sequences and monoclonal antibodies. Here, we sought to characterise a third class of αvβ6-targeted agent comprising the knob domain of the Ad5NULLA20 oncolytic virotherapy platform.
Through incorporation of S408E, P409A substitution mutations (collectively referred to as KO1) that ablate native viral binding to the coxsackie and adenovirus receptor and insertion of a 20 amino acid (A20) sequence into the flexible HI loop, the trimeric C-terminal knob domain of the Ad5NULLA20 fiber protein has been retargeted to bind αvβ6 with exquisite specificity (2). Recombinant N-terminally his-tagged versions of this domain were purified from E.coli and crystallised. A high resolution (1.27Å) structure was generated which clearly revealed the  presence of the KO1 mutation. However, the high flexibility of the A20 insert precluded resolution of this region in the crystal structure. Small angle X-ray scattering results were nevertheless consistent with this forming a protrusion extending away from the trimer core as indicated by small increases in Rg and Dmax values compared to the wildtype knob domain, consistent with the conformation predicted by homology modelling.
In order to investigate the in vivo biodistributions of the different classes of αvβ6-targeted agents, the Ad5.KO1.A20 knob domain, along with an anti-αvβ6 monoclonal antibody (620W.7) and A20 peptide sequence were subsequently conjugated to the long half-life radiotracer Zr89. Longitudinal micro-PET imaging of CD1 nude mice bearing matched αvβ6-positive A375-β6 and αvβ6-negative A375 tumours on opposite hips revealed selective trafficking of 620W.7 and a streptavidin-biotin-A20 peptide complex to tumours expressing this integrin. In contrast, rapid bowel-mediated excretion of Ad5.KO1.A20 was observed, with minimal accumulation in αvβ6-expressing tumours.
These results offer novel insights into the structural and biological features of different classes of αvβ6-targeted biologics that may have potential as diagnostic imaging or selective drug delivery agents.

References
1. Brzozowska, E. et al. 2022. Integrin Alpha v Beta 6 (αvβ6) and Its Implications in Cancer Treatment. Int J Mol Sci 23(20),  doi: 10.3390/ijms232012346
2. Uusi-Kerttula, H. et al. 2018. Ad5(NULL)-A20: A Tropism-Modified, αvβ6 Integrin-Selective Oncolytic Adenovirus for Epithelial Ovarian Cancer Therapies. Clin Cancer Res 24(17), pp. 4215-4224. doi: 10.1158/1078-0432.Ccr-18-1089

Development of Novel enzymes for increased activity by directed evolution using microfluidic approaches

Hayley Blaber – University of Exeter

Two novel hydrolases, identified through bioinformatic analysis of thermophilic sequence databases such as Hotzyme, have been characterised and shown to exhibit activity against multiple substrates, under a range of conditions. Using metagenomic databases allows the discovery of enzymes from organisms that have not been, or cannot be cultured1.
Using error-prone PCR, libraries of approximately 100,000 gene variants were created for each enzyme. The enzyme variants were screened for activity using microfluidic water-in-oil droplets containing individual cells expressing the enzyme. The cells are combined with a lysis agent and a fluorogenic substrate before screening using a second microfluidic device. A laser is used to excite the fluorescent product which is detected so that droplets exhibiting higher fluorescence, and likely higher enzyme activity, can be sorted2.
Using microfluidic techniques for screening and sorting, a high volume of variants can be screened in a single experiment, giving a great advantage over traditional techniques used in directed evolution3. The high throughput of this method gives a better chance of discovering successful variants4.
Data collected from the screening indicates that a number of variants exhibited significantly higher activity than the wild type enzymes. Further steps include expression and characterisation of hits to determine the overall success of the approach.

References
1. Ferrer, M., Martínez-Martínez, M., Bargiela, R., Streit, W. R., Golyshina, O. V., & Golyshin, P. N. (2016). Estimating the success of enzyme bioprospecting through metagenomics: Current status and future trends. Microbial Biotechnology, 9: 22–34,
2. Scherli, Y. and Hollfelder, F. (2009) The potential of microfluidic water-in-oil droplets in experimental biology. Molecular BioSystems. 5:1392-1404
3. Yu, T., Lane, ST., Zhao, H (2021). Directed evolution: Methodologies and applications. Chemical reviews. 121:12384-12444,
4. Agresti, J. J., Antipov, E., Abate, A. R., Ahn, K., Rowat, A. C., Baret, J. C., Marquez, M., Klibanov, A. M., Griffiths, A. D., & Weitz, D. A. (2010).Ultrahigh-throughput screening in drop-based microfluidics for directed evolution Proceedings of the National Academy of Sciences of the United States of America, 107: 6560.

Using integrative structural biology to develop more powerful immunostimulatory antibodies for cancer immunotherapy.

Isabel Elliott – University of Southampton

Immunostimulatory antibodies (ISAs) represent a promising strategy for cancer immunotherapy. By binding and activating co-stimulatory molecules, such as certain tumour necrosis factor receptors (TNFRs), expressed on immune cells, ISAs can enhance immune responses towards tumours, resulting in powerful anti-cancer effects (1). However, to develop more effective therapeutics, we need to gain a deeper understanding of the structure-function relationship behind ISA agonistic activity.
Antibodies comprise two identical antigen-binding (F(ab)) domains linked to an effector (Fc) domain through a hinge containing several disulfides. There are four isotypes of human IgG, and previous work has shown that human (h)IgG2 antibodies can deliver strong agonistic activity for ISAs, due to their unique hinge disulfide arrangement (2). Using a series of hIgG2 cysteine to serine exchange mutations in the hinge of a clinically-relevant ISA targeting the TNFR CD40, it was found that strong agonistic activity was associated with the presence of a disulfide cross-over formation in the hinge. This cross-over was shown to restrict global antibody flexibility and reduce conformational sampling in solution (3).
To extend these findings, we sought to investigate whether further restriction of the hinge via the introduction of additional mutations would provide augmented agonism in different human antibody isotypes. New antibody variants were characterised in a host of biophysical, biochemical and cellular assays (SDS-PAGE, CE-SDS, NFκB-GFP reporters, flow cytometry and surface plasmon resonance) to ascertain their binding properties and activity. Subsequently, X-ray crystallography, including sulfur single wavelength anomalous diffraction, was performed to determine high-resolution atomic structures. Flexibility and conformation were then assessed using small angle X-ray scattering (SAXS). This structural information was integrated with molecular dynamics simulations to characterise the link between ISA conformational dynamics/flexibility and agonistic activity. Together, these orthogonal and complementary approaches provide a rational means to develop more powerful ISAs to deliver more effective anti-cancer treatments.
(1) Weiner, L. M., Murray, J. C., and Shuptrine, C. W. (2012) Antibody-Based Immunotherapy of Cancer. Cell 148, 1081-1084
(2) White, A. L., Chan, H. T. C., French, R. R., Willoughby, J., Mockridge, C. I., Roghanian, A., Penfold, C. A., Booth, S. G., Dodhy, A., Polak, M. E., Potter, E. A., Ardern-Jones, M. R., Verbeek, J. S., Johnson, P. W. M., Al-Shamkhani, A., Cragg, M. S., Beers, S. A., and Glennie, M. J. (2015) Conformation of the Human Immunoglobulin G2 Hinge Imparts Superagonistic Properties to Immunostimulatory Anticancer Antibodies. Cancer Cell 27, 138-148
(3) Orr, C. M., Fisher, H., Yu, X., Chan, C. H., Gao, Y., Duriez, P. J., Booth, S. G., Elliott, I., Inzhelevskaya, T., Mockridge, C. I., Penfold, C. A., Wagner, A., Glennie, M. J., White, A. L., Essex, J. W., Pearson, A. R., Cragg, M. S., and Tews, I. (2022) Hinge disulfides in human IgG2 CD40 antibodies modulate receptor signaling by regulation of conformation and flexibility. Science Immunology 7, eabm3723

Droplet microfluidics for time-resolved serial crystallography

Jack Stubbs – University of Southampton and Diamond Light Source

Jack Stubbs a,b, Theo Hornsey a, Niall Hanrahan c,d, Rachel Bolton a,b, Shibom Basu e, Julien Orlans e, Daniele de Sanctis e, Martin Maly a, Luis Blay-Esteban f, Jung-uk Shim g, Patrick D. Shaw Stewart h, Allen Orville b,i, Ivo Tews a,d, Jonathan West d,j
a School of Biological Sciences, Faculty of Environmental and Life Sciences, University of Southampton, SO17 1BJ, United Kingdom
b Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, United Kingdom
c School of Chemistry, Faculty of Engineering and Physical Sciences, University of Southampton, SO17 1BJ, United Kingdom
d Institute for Life Sciences, University of Southampton, SO17 1BJ, United Kingdom
e ESRF The European Synchrotron, 71 Avenue des Martyrs, 38042 Grenoble Cedex 9, France
f Universitat Carlemany, Av. Verge de Canolich, 47, AD600 Sant Julia de Loria, Principat d’Andorra, Spain
g School of Physics and Astronomy, University of Leeds, Leeds LS2 9JT, United Kingdom
h Douglas Instruments Ltd, East Garston, Hungerford RG17 7HD, United Kingdom
i Research Complex at Harwell, Harwell Science and Innovation Campus, Didcot, OX11 0FA, United Kingdom
j Cancer Sciences, Faculty of Medicine, University of Southampton, SO17 1BJ, United Kingdom
The future of time-resolved protein crystallography will depend on the availability of large numbers of uniformly small microcrystals, rapid mixing for the addition of substrates and frequency matching with the beam for efficient throughput. Here we report the use of droplet microfluidics to effect volumetric scaling for the production of uniform 3 mm lysozyme crystals and extend the approach to the production of uniform 2 mm Pdx1 crystals using a seeding strategy to overcome the nucleation improbability. Droplets were also explored as vehicles for mixing crystals with substrates and frequency modulation to synchronise with beam sources. Mixing times scale with droplet volume and velocity, achieving millisecond mixing at frequencies suitable for interfacing with beam lines. Microchannels fabricated from x-ray and electron transmissible materials are advocated for frequency-matched time-resolved experiments involving synchrotron and XFEL analysis modalities.

Tools for serial crystallography in CCP4: current state and future plans

Martin Malý – University of Southampton

The development of X-ray radiation sources – mainly availability of the X-ray free electron lasers – leads to increased interest in serial macromolecular crystallography (serial MX). This method allows us to perform e.g. time-resolved studies, collection of data not affected by radiation damage, and pump and probe experiments – all at room temperature. These new approaches make new challenges in sample preparation, diffraction experiment and data processing.
Software tools for serial MX currently available in the CCP4 suite will be presented. Moreover, possible directions of future development will be discussed with a focus on the analysis of differences between series of data sets in real and reciprocal space.

Studies on α-methylacyl-CoA racemase

Otsile Mojanaga – University of Bath

Prostate cancer is the most diagnosed male cancer, with 1.3 million new cases annually and the second highest mortality rate among men which makes its a disease of global concern (Islami et al., 2021).  α-Methylacyl-CoA racemase (AMACR;P504S; EC 5..99.4) is over produced by PCa cells and its knowdown or inhibition has been shown to convert androgen-independent cells to their androgen-dependent state. Consequently, hAMACR is potential PCa biomarker and drug target. Rationally designed hAMACR inhibitors have been reported, but further development of new inhibitors has been impeded by the lack of a hAMACR structure. Mycobacterium tuberculosis α-Methylacyl-CoA racemase (MCR) has a 44% sequence identity with hAMMCR, has a protein structure and is a model for the hAMACR enzyme (Bhaumik et al., 2007)

References
Bhaumik, P., Schmitz, W., Hassinen, A., Hiltunen, J. K., Conzelmann, E., & Wierenga, R. K. (2007). The catalysis of the 1,1-proton transfer by α-methyl-acyl-CoA racemase is coupled to a movement of the fatty acyl moiety over a hydrophobic, methionine-rich surface. Journal of Molecular Biology, 367(4), 1145–1161. https://doi.org/10.1016/J.JMB.2007.01.062
Islami, F., Ward, E. M., Sung, H., Cronin, K. A., Tangka, F. K. L., Sherman, R. L., Zhao, J., Anderson, R. N., Henley, S. J., Yabroff, K. R., Jemal, A., & Benard, V. B. (2021). Annual report to the nation on the status of cancer, part 1: National cancer statistics. JNCI: Journal of the National Cancer Institute, 113(12), 1648–1669. https://doi.org/10.1093/JNCI/DJAB131
Lloyd, M. D., Yevglevskis, M., Lee, G. L., Wood, P. J., Threadgill, M. D., & Woodman, T. J. (2013). α-Methylacyl-CoA racemase (AMACR): Metabolic enzyme, drug metabolizer and cancer marker P504S. Progress in Lipid Research, 52(2), 220–230. https://doi.org/10.1016/J.PLIPRES.2013.01.001
Yevglevskis, M., Lee, G. L., Nathubhai, A., Petrova, Y. D., James, T. D., Threadgill, M. D., Woodman, T. J., & Lloyd, M. D. (2017). A novel colorimetric assay for α-methylacyl-CoA racemase 1A (AMACR; P504S) utilizing the elimination of 2,4-dinitrophenolate. Chemical Communications, 53(37), 5087–5090. https://doi.org/10.1039/C7CC00476A

Structural characterisation of the novel cyclic 2,3-diphosphoglycerate synthetase from Methanothermus fervidus involved in extremolyte synthesis

Simone De Rose – University of Exeter

Simone A. De Rose1, Michail N. Isupov1, Harley L. Worthy2, Nicholas J. Harmer3, Jennifer A. Littlechild1
1)The Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental Sciences, University of Exeter, United Kingdom 
2) Geoffrey Pope Building, Biosciences, College of Life and Environmental Sciences, University of Exeter, United Kingdom 
3)Living Systems Institute, University of Exeter, United Kingdom
E-mail: s.a.de-rose@exeter.ac.uk
Cyclic 2,3 -diphosphoglycerate is an extremolyte found exclusively in the hyperthermophilic archaeal methanogens, such as Methanothermus fervidus, Methanopyrus kandleri, and Methanothermobacter thermoautotrophicus. Cyclic 2,3 -diphosphoglycerate has been proven to increase the thermostability of proteins and to protect DNA against oxidative damage caused by hydroxyl radicals. The extremolyte is biosynthesized by a two-step pathway from the glycolytic intermediate 2-phosphoglycerate. The process requires two enzymes. 2-phosphoglycerate kinase which forms 2,3-di-phosphoglycerate from 2-phosphoglycerate, and cyclic di-phosphoglycerate synthase which cyclizes 2,3 -diphosphoglycerate to form the extremolyte cyclic 2,3 -diphosphoglycerate.
The cyclic 2,3-diphosphoglycerate synthetase enzyme has been crystallised and its structure solved to 1.7 Å resolution. It has also been crystallised in complex with its substrate 2,3 diphosphoglycerate and the co-factor ADP and this structure has been solved to 2.2 Å resolution. The enzyme structure has three domains, two of which have a unique fold. The core domain fold has some similarity with other NTP-dependent enzymes.  A large percentage of the structure, including a 127 amino acid region at the N-terminus has no structural similarity to other known enzyme structures. The structures of the complex show a large conformational change that occurs in the enzyme structure during its turnover. This involves the transfer of the γ-phosphate group from ATP to the substrate 2,3-di-phosphoglycerate which then continues with a SN2 attack to form a phosphoanhydride resulting in the unusual cyclic 2,3 -diphosphoglycerate extremolyte.

Structure Function Analysis of Greatwall

William Foster – University of Sussex

Microtubule-associated serine/threonine kinase like (MASTL), also commonly known as Greatwall (Gwl), is an essential protein for the normal regulation and progression of mitosis. Through phosphorylating its substrates ENSA and ARPP19, Gwl leads to the inhibition of protein phosphatase 2A (PP2A). This inactivation of PP2A allows for the activation of a major mitotic kinase, cyclin-dependent kinase 1 (CDK1) and the phosphorylation of its many substrates, leading to mitotic progression. As a kinase, Gwl consists of the expected N and C lobes conserved throughout this class of protein. However, it possesses a unique and substantial ~500 amino acid insertion within these core domains known as the Non-Conserved Middle Region (NCMR). With limited studies and structural data missing for this large region of the protein, its role remains unclear. Nevertheless, with Gwl emerging as a potentially promising target in cancer therapy, it is crucial this unknown is studied. This project will aim to build upon the solved kinase domain structure of Gwl, with the goal of elucidating the full-length form using cryo-electron microscopy. Alongside this I will work with recombinantly expressed protein to test and aid in the development of Gwl specific inhibitors, while also using live imaging of mammalian cell lines for functional studies.

Breaking Bad Bugs: Cooking Up a Cure For Coxiella burnetii

William Stuart – University of Exeter

Coxiella burnetii is a highly pathogenic zoonotic bacterium. It causes the Q-Fever in humans. Ruminants are the main animal reservoirs and previous outbreaks resulted in significant economic damage. Farm and veterinary workers are at highest risk, with infections in the wider community likely underreported. There is no human vaccine available outside Australia. Current therapy for chronic infections relies on a long antibiotic course (at least 18 months). This work will produce a prioritised list of targets for drug discovery and demonstrate feasibility by generating initial hits in order to aid the development of a targeted therapeutic against Coxiella burnetii. We developed a pipeline to assess essential C. burnetii genes and have progressed a candidate to the initial steps of structure based drug design utilising X-ray crystallography.

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No abstract but poster:

Hayden Fisher

Daniel Burns

Darwin Tu

Abigail Talbot