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Abstract: Coxsackievirus A24 variant (CVA24v) is the primary causative agent of the highly contagious eye infection designated acute hemorrhagic conjunctivitis (AHC). It is solely responsible for two pandemics and several recurring outbreaks of the disease over the last decades, thus affecting millions of individuals throughout the world. To date, no antiviral agents or vaccines are available for combating this disease, and treatment is mainly supportive. CVA24v utilizes Neu5Ac-containing glycans as attachment receptors facilitating entry into host cells. We have previously reported that pentavalent Neu5Ac conjugates based on a glucose-scaffold inhibit CVA24v infection of human corneal epithelial cells. In this study, we report on the design and synthesis of scaffold-replaced pentavalent Neu5Ac conjugates and their effect on CVA24v cell transduction and the use of cryogenic electron microscopy (cryo-EM) to study the binding of these multivalent conjugates to CVA24v. The results presented here provide insights into the development of Neu5Ac-based inhibitors of CVA24v and, most significantly, the first application of cryo-EM to study the binding of a multivalent ligand to a lectin.

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Abstract: Pseudomonas are a common cause of hospital-acquired infections that may be lethal. ADP-ribosyltransferase activities of Pseudomonas exotoxin-S and -T depend on 14-3-3 proteins inside the host cell. By binding in the 14-3-3 phosphopeptide binding groove, an amphipathic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3β:ExoS and -ExoT complexes presented here reveal an extensive hydrophobic interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the amphipathic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the amphipathic C-terminus with a fragment from Vibrio Vis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, we show that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the amphipathic C-terminal segment.

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Abstract: Inhibiting ADP-ribosyl transferases with PARP-inhibitors is considered a promising strategy for the treatment of many cancers and ischemia, but most of the cellular targets are poorly characterized. Here, we describe an inhibitor of ADP-ribosyltransferase-3/poly(ADP-ribose) polymerase-3 (ARTD3), a regulator of DNA repair and mitotic progression. In vitro profiling against 12 members of the enzyme family suggests selectivity for ARTD3, and crystal structures illustrate the molecular basis for inhibitor selectivity. The compound is active in cells, where it elicits ARTD3-specific effects at submicromolar concentration. Our results show that by targeting the nicotinamide binding site, selective inhibition can be achieved among the closest relatives of the validated clinical target, ADP-ribosyltransferase-1/poly(ADP-ribose) polymerase-1.

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Abstract: Yersinia pseudotuberculosis is a Gram-negative bacterium that is capable of causing a tuberculosis-like disease in humans and animals, as well as being a model organism for Y. pestis (Robins-Browne & Hartland, 2003[Robins-Browne, R. & Hartland, E. (2003). International Handbook of Foodborne Pathogens, edited by M. D. Miliotis & J. W. Bier, pp. 323-356. New York: Marcel Dekker.]). Thiol peroxidase (Tpx; p20; UniProt accession No. Q66A71) is an atypical 2-Cys peroxiredoxin that uses the redox potential from thioredoxin reductase and thioredoxin I to reduce alkyl hydroperoxides (Baker & Poole, 2003[Baker, L. M. & Poole, L. B. (2003). J. Biol. Chem. 278, 9203-9211.]). Tpx was initially suggested to be localized to the periplasm and to be involved in the removal of lipid hydroxyperoxides produced by oxidative stress (Cha et al., 1995[Cha, M.-K., Kim, H.-K. & Kim, I.-H. (1995). J. Biol. Chem. 270, 28635-28641.]). However, recent studies of fractionated cells show that Tpx is cytoplasmic and is released into the periplasm as a response to stress (Tao, 2008[Tao, K. (2008). FEMS Microbiol. Lett. 289, 41-45.]). Tpx contains three cysteines, two of which (Cys61 and Cys95, with Cys95 being the resolving cysteine) form the redox-active pair (Baker & Poole, 2003[Baker, L. M. & Poole, L. B. (2003). J. Biol. Chem. 278, 9203-9211.]). Despite the presence of a third cysteine, there is no covalent dimerization in the oxidized state as is observed for most 2-Cys peroxiredoxins (Baker & Poole, 2003[Baker, L. M. & Poole, L. B. (2003). J. Biol. Chem. 278, 9203-9211.]). The structure of Tpx from Escherichia coli has been elucidated in the oxidized form and the mutated C61S form (Hall et al., 2009[Hall, A., Sankaran, B., Poole, L. B. & Karplus, P. A. (2009). J. Mol. Biol. 393, 867-881.]; Choi et al., 2003[Choi, J., Choi, S., Cha, M.-K., Kim, I.-H. & Shin, W. (2003). J. Biol. Chem. 278, 49478-49486.]) and shows a sequence identity of approximately 80% to Y. pseudotuberculosis Tpx (ypTpx). Here, we describe the crystallization of ypTpx in three different crystal forms grown under different conditions, as well as the crystallization of the catalytically inactive (Baker & Poole, 2003[Baker, L. M. & Poole, L. B. (2003). J. Biol. Chem. 278, 9203-9211.]) mutant ypTpxC61S.