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Finding out about sample preparation and transportation of structural biology samples in Acta Crystallographica F, Structural Biology Communications.
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Manejo de Especímenes , Cristalografía por Rayos X , Manejo de Especímenes/métodos , HumanosRESUMEN
Background: Endolysins are phage-encoded lytic enzymes that degrade bacterial peptidoglycan at the end of phage lytic cycles to release new phage particles. These enzymes are being explored as an alternative to small-molecule antibiotics. Methods: The crystal structure of KTN6 Gp46 was determined and compared with a ColabFold model. Cleavage specificity was examined using a peptidoglycan digest and reversed-phase high-performance liquid chromatography coupled to mass spectrometry (HPLC/MS). Results: The structure of KTN6 Gp46 could be determined at 1.4 Å resolution, and key differences in loops of the putative peptidoglycan binding domain were identified in comparison with its closest known homologue, the endolysin of phage SPN1S. Reversed-phase HPLC/MS analysis of the reaction products following peptidoglycan digestion confirmed the muramidase activity of Gp46, consistent with structural predictions. Conclusion: These insights into the structure and function of endolysins further expand the toolbox for endolysin engineering and explore their potential in enzyme-based antibacterial design strategies.
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The Acta Cryst. F - Structural Biology Communications Editors explain how important international collaborations are in science and structural biology.
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Cooperación Internacional , Publicaciones Periódicas como Asunto , HumanosRESUMEN
The current situation of scientific manuscript peer review is discussed, both generally and as applied to Acta Crystallographica F - Biological Research Communications.
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Revisión por Pares , Cristalografía por Rayos XRESUMEN
Acta Cryst. F - Structural Biology Communications plans to introduce more video content to articles and Dialpuri et al. [(2024). Acta Cryst. F80, 30-35] provide an early example.
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Polisacáridos , Cristalografía por Rayos XRESUMEN
Mark van Raaij introduces the two new Section Editors who have been appointed to Structural Biology Communications - Acta Cryst. F.
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Biología , Cristalografía por Rayos XRESUMEN
This editorial acknowledges the transformative impact of new machine-learning methods, such as the use of AlphaFold, but also makes the case for the continuing need for experimental structural biology.
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Biología , Aprendizaje Automático , Microscopía por Crioelectrón , Cristalografía por Rayos XRESUMEN
This editorial acknowledges the transformative impact of new machine-learning methods, such as the use of AlphaFold, but also makes the case for the continuing need for experimental structural biology.
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Biología , Microscopía por Crioelectrón , Cristalografía por Rayos X , Conformación ProteicaRESUMEN
This editorial acknowledges the transformative impact of new machine-learning methods, such as the use of AlphaFold, but also makes the case for the continuing need for experimental structural biology.
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Biología , Aprendizaje Automático , Microscopía por Crioelectrón , Cristalografía por Rayos X , Conformación ProteicaRESUMEN
Irreversible inhibition of the enzyme type I dehydroquinase (DHQ1), a promising target for anti-virulence drug development, has been explored by enhancing the electrophilicity of specific positions of the ligand towards covalent lysine modification. For ligand design, we made use of the advantages offered by the intrinsic acid-base properties of the amino substituents introduced in the quinate scaffold, namely compounds 6-7 (R configuration at C3), to generate a potential leaving group, as well as the recognition pattern of the enzyme. The reactivity of the C2-C3 bond (Re face) in the scaffold was also explored using compound 8. The results of the present study show that replacement of the C3 hydroxy group of (-)-quinic acid by a hydroxyamino substituent (compound 6) provides a time-dependent irreversible inhibitor, while compound 7, in which the latter functionality was substituted by an amino group, and the introduction of an oxirane ring at C2-C3 bond, compound 8, do not allow covalent modification of the enzyme. These outcomes were supported by resolution of the crystal structures of DHQ1 from Staphylococcus aureus (Sa-DHQ1) and Salmonella typhi (St-DHQ1) chemically modified by 6 at a resolution of 1.65 and 1.90 Å, respectively, and of St-DHQ1 in the complex with 8 (1.55 Å). The combination of these structural studies with extensive molecular dynamics simulation studies allowed us to understand the molecular basis of the type of inhibition observed. This study is a good example of the importance of achieving the correct geometry between the reactive center of the ligand (electrophile) and the enzyme nucleophile (lysine residue) to allow selective covalent modification. The outcomes obtained with the hydroxyamino derivative 6 also open up new possibilities in the design of irreversible inhibitors based on the use of amino substituents.
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Wall teichoic acids (WTAs) are glycopolymers decorating the surface of Gram-positive bacteria and potential targets for antibody-mediated treatments against Staphylococcus aureus, including methicillin-resistant (MRSA) strains. Through a combination of glycan microarray, synthetic chemistry, crystallography, NMR, and computational studies, we unraveled the molecular and structural details of fully defined synthetic WTA fragments recognized by previously described monoclonal antibodies (mAbs 4461 and 4497). Our results unveiled the structural requirements for the discriminatory recognition of α- and ß-GlcNAc-modified WTA glycoforms by the complementarity-determining regions (CDRs) of the heavy and light chains of the mAbs. Both mAbs interacted not only with the sugar moiety but also with the phosphate groups as well as residues in the ribitol phosphate (RboP) units of the WTA backbone, highlighting their significant role in ligand specificity. Using elongated WTA fragments, containing two sugar modifications, we also demonstrated that the internal carbohydrate moiety of α-GlcNAc-modified WTA is preferentially accommodated in the binding pocket of mAb 4461 with respect to the terminal moiety. Our results also explained the recently documented cross-reactivity of mAb 4497 for ß-1,3/ß-1,4-GlcNAc-modified WTA, revealing that the flexibility of the RboP backbone is crucial to allow positioning of both glycans in the antibody binding pocket.
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Phage lysins are a source of novel antimicrobials to tackle the bacterial antibiotic-resistance crisis. The engineering of phage lysins is being explored as a game-changing technological strategy to introduce a more precise approach in the way in which antimicrobial therapy is applied. Such engineering efforts will benefit from a better understanding of lysin structure and function. In this work, the antimicrobial activity of the endolysin from Pseudomonas aeruginosa phage JG004, termed Pae87, has been characterized. This lysin had previously been identified as an antimicrobial agent candidate that is able to interact with the Gram-negative surface and disrupt it. Further evidence is provided here based on a structural and biochemical study. A high-resolution crystal structure of Pae87 complexed with a peptidoglycan fragment showed a separate substrate-binding region within the catalytic domain, 18â Å away from the catalytic site and located on the opposite side of the lysin molecule. This substrate-binding region was conserved among phylogenetically related lysins lacking an additional cell-wall-binding domain, but not among those containing such a module. Two glutamic acids were identified to be relevant for the peptidoglycan-degradation activity, although the antimicrobial activity of Pae87 was seemingly unrelated. In contrast, an antimicrobial peptide-like region within the Pae87 C-terminus, named P87, was found to be able to actively disturb the outer membrane and display antibacterial activity by itself. Therefore, an antimicrobial mechanism for Pae87 is proposed in which the P87 peptide plays the role of binding to the outer membrane and disrupting the cell-wall function, either with or without the participation of the catalytic activity of Pae87.
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Bacteriófagos , Fagos Pseudomonas , Péptidos Antimicrobianos , Bacteriófagos/metabolismo , Muramidasa/metabolismo , Pseudomonas aeruginosaRESUMEN
The editors discuss the submission of structural biology data.
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Artefactos , Cristalografía por Rayos X/normas , Proteínas de Escherichia coli/ultraestructura , Escherichia coli/genética , Hidroliasas/ultraestructura , Transgenes , Cianatos/química , Cianatos/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Humanos , Hidroliasas/genética , Hidroliasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Serratia/genética , Serratia/metabolismoRESUMEN
Disabling the bacterial capacity to cause infection is an innovative approach that has attracted significant attention to fight against superbugs. A relevant target for anti-virulence drug discovery is the type I dehydroquinase (DHQ1) enzyme. It was shown that the 2-hydroxyethylammonium derivative 3 has in vitro activity since it causes the covalent modification of the catalytic lysine residue of DHQ1. As this compound does not bear reactive electrophilic centers, how the chemical modification occurs is intriguing. We report here an integrated approach, which involves biochemical studies, X-ray crystallography and computational studies on the reaction path using combined quantum mechanics/molecular mechanics Umbrella Sampling Molecular Dynamics, that evidences that DHQ1 catalyzes its self-immolation by transforming the unreactive 2-hydroxyethylammonium group in 3 into an epoxide that triggers the lysine covalent modification. This finding might open opportunities for the design of lysine-targeted irreversible inhibitors bearing a 2-hydroxyethylammonium moiety as an epoxide proform, which to our knowledge has not been reported previously.
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Bacterias/química , Inhibidores Enzimáticos/química , Compuestos Epoxi/química , Hidroliasas/química , Bacterias/metabolismo , Catálisis , Descubrimiento de Drogas , Hidroliasas/metabolismo , Lisina , Simulación de Dinámica MolecularRESUMEN
Tailed bacteriophages (phages) are one of the most abundant life forms on Earth. They encode highly efficient molecular machines to infect bacteria, but the initial interactions between a phage and a bacterium that then lead to irreversible virus attachment and infection are poorly understood. This information is critically needed to engineer machines with novel host specificities in order to combat antibiotic resistance, a major threat to global health today. The tailed phage T4 encodes a specialized device for this purpose, the long tail fiber (LTF), which allows the virus to move on the bacterial surface and find a suitable site for infection. Consequently, the infection efficiency of phage T4 is one of the highest, reaching the theoretical value of 1. Although the atomic structure of the tip of the LTF has been determined, its functional architecture and how interactions with two structurally very different Escherichia coli receptor molecules, lipopolysaccharide (LPS) and outer membrane protein C (OmpC), contribute to virus movement remained unknown. Here, by developing direct receptor binding assays, extensive mutational and biochemical analyses, and structural modeling, we discovered that the ball-shaped tip of the LTF, a trimer of gene product 37, consists of three sets of symmetrically alternating binding sites for LPS and/or OmpC. Our studies implicate reversible and dynamic interactions between these sites and the receptors. We speculate that the LTF might function as a "molecular pivot" allowing the virus to "walk" on the bacterium by adjusting the angle or position of interaction of the six LTFs attached to the six-fold symmetric baseplate.