Crystallization Of The CHAP Domain Of The Endolysin From Staphylococcus Aureus Bacteriophage K

CHAPK is the N-terminal cysteine, histidine-dependent amidohydrolase/peptidase domain (CHAP domain) of the Staphylococcus aureus bacteriophage K endolysin LysK. It is formed from the first 165 residues of LysK and functions by cleaving specific peptidoglycan peptide bonds, causing bacterial lysis. CHAPK can lyse S. aureus when applied exogenously, making it a good candidate for the treatment of multidrug-resistant Staphylococcus aureus infections. Here, the crystallization of CHAPK and the collection of native and derivative data to high resolution, which allowed structure solution, are reported. The structure may help to elucidate the mechanism of action and in the design of chimeric proteins or mutants with improved antibacterial activity

Inhibition of L. monocytogenes Biofilm Formation by the Amidase Domain of the Phage vB_LmoS_293 Endolysin

peer-reviewedListeria monocytogenes is a ubiquitous Gram-positive bacterium that is a major concern for food business operators because of its pathogenicity and ability to form biofilms in food production environments. Bacteriophages (phages) have been evaluated as biocontrol agents for L. monocytogenes in a number of studies and, indeed, certain phages have been approved for use as anti-listerial agents in food processing environments (ListShield and PhageGuard Listex). Endolysins are proteins produced by phages in the host cell. They cleave the peptidoglycan cell wall, thus allowing release of progeny phage into the environment. In this study, the amidase domain of the phage vB_LmoS_293 endolysin (293-amidase) was cloned and expressed in Escherichia. coli(E. coli). Muralytic activity at different concentrations, pH and temperature values, lytic spectrum and activity against biofilms was determined for the purified 293-amidase protein. The results showed activity on autoclaved cells at three different temperatures (20 °C, 37 °C and 50 °C), with a wider specificity (L. monocytogenes 473 and 3099, a serotype 4b and serogroup 1/2b-3b-7, respectively) compared to the phage itself, which targets only L. monocytogenes serotypes 4b and 4e. The protein also inhibits biofilm formation on abiotic surfaces. These results show the potential of using recombinant antimicrobial proteins against pathogens in the food production environment

Hydroxylammonium derivatives for selective active-site lysine modification in the anti-virulence bacterial target DHQ1 enzyme

Targeted irreversible inhibitors bearing electrophiles that become activated towards covalent bond formation upon binding to a specific protein/enzyme is an emerging area in drug discovery. Targeting lysine residues is challenging due to the intrinsically low reactivity of the amino group at physiological pH. Herein we report the first example of a hydroxylammonium derivative that causes a specific covalent modification of an active-site and a sterically inaccessible lysine residue of an enzyme. The described ligands, compounds 1–3, were rationally designed to be activated towards covalent bond formation upon binding to the type I dehydroquinase (DHQ1) enzyme for the development of new anti-virulence agents to combat the widespread resistance to antibiotics. Evidence in atomic detail for the covalent modifications caused by the ligands to the catalytic Lys170 by the formation of a stable secondary amine is provided by the resolution at 1.08–1.25 Å of the crystal structures of DHQ1 from Salmonella typhi enzyme adducts. In addition, the first crystal structure of the addition intermediate adduct at 1.4 Å of a Schiff base formation reaction by using an analog of the natural substrate, compound 4, is also reported. Molecular dynamics simulation studies on non-covalent enzyme/ligand complexes and a two-dimensional QM/MM umbrella sampling simulation study suggested that a direct displacement by Lys170 with the release of NH2OH would be feasible. These studies might open up new opportunities for the development of novel lysine-targeted irreversible inhibitors bearing a methylhydroxylammonium moiety as a latent electrophile.Financial support from the Spanish Ministry of Economy and Competiveness (SAF2016-75638-R), the Xunta de Galicia [Centro singular de investigación de Galicia accreditation 2016-2019 (ED431G/09) and ED431B 2018/04] and theEuropean Union (European Regional Development Fund –ERDF) is gratefully acknowledged. MM and EL thank the Spanish Ministry of Education, Culture and Sport and the Xunta de Galicia for their respective FPU and postdoctoral fellowshipsS

The Eighth Central European Conference "Chemistry towards Biology": snapshot

The Eighth Central European Conference "Chemistry towards Biology" was held in Brno, Czech Republic, on 28 August – 1 September 2016The Eighth Central European Conference "Chemistry towards Biology" was held in Brno, Czech Republic, on 28 August-1 September 2016 to bring together experts in biology, chemistry and design of bioactive compounds; promote the exchange of scientific results, methods and ideas; and encourage cooperation between researchers from all over the world. The topics of the conference covered "Chemistry towards Biology", meaning that the event welcomed chemists working on biology-related problems, biologists using chemical methods, and students and other researchers of the respective areas that fall within the common scope of chemistry and biology. The authors of this manuscript are plenary speakers and other participants of the symposium and members of their research teams. The following summary highlights the major points/topics of the meeting

Crystallographic structure determination of bacteriophage endolysins

Bacteriophages produce endolysins that target and cleave the hosts peptidoglycan to release their progeny at the end of the infection cycle. These proteins can be used for the eradication of pathogenic bacteria, but also for their detection. Endolysins may contain a single catalytic domain or several domains, including a cell wall binding domain. To understand their function in detail and design mutated or chimeric molecules with novel properties, knowledge of their structures and detailed mechanisms is necessary. X-ray protein crystallography is an excellent method to obtain high-resolution structures of biological macromolecules, and here we describe the method and the folds of known endolysin domains.This work was supported by grant BFU2017-82207-P from the Spanish Ministry of Science and Innovation to MJvR and by a RISAM PhD fellowship from the Cork Institute of Technology to MS-G

Crystal structure of the lytic CHAPK domain of the endolysin LysK from Staphylococcus aureus bacteriophage K

[Background] Bacteriophages encode endolysins to lyse their host cell and allow escape of their progeny. Endolysins are also active against Gram-positive bacteria when applied from the outside and are thus attractive anti-bacterial agents. LysK, an endolysin from staphylococcal phage K, contains an N-terminal cysteine-histidine dependent amido-hydrolase/peptidase domain (CHAPK), a central amidase domain and a C-terminal SH3b cell wall-binding domain. CHAPK cleaves bacterial peptidoglycan between the tetra-peptide stem and the penta-glycine bridge.[Methods] The CHAPK domain of LysK was crystallized and high-resolution diffraction data was collected both from a native protein crystal and a methylmercury chloride derivatized crystal. The anomalous signal contained in the derivative data allowed the location of heavy atom sites and phase determination. The resulting structures were completed, refined and analyzed. The presence of calcium and zinc ions in the structure was confirmed by X-ray fluorescence emission spectroscopy. Zymogram analysis was performed on the enzyme and selected site-directed mutants.[Results] The structure of CHAPK revealed a papain-like topology with a hydrophobic cleft, where the catalytic triad is located. Ordered buffer molecules present in this groove may mimic the peptidoglycan substrate. When compared to previously solved CHAP domains, CHAPK contains an additional lobe in its N-terminal domain, with a structural calcium ion, coordinated by residues Asp45, Asp47, Tyr49, His51 and Asp56. The presence of a zinc ion in the active site was also apparent, coordinated by the catalytic residue Cys54 and a possible substrate analogue. Site-directed mutagenesis was used to demonstrate that residues involved in calcium binding and of the proposed active site were important for enzyme activity.[Conclusions] The high-resolution structure of the CHAPK domain of LysK was determined, suggesting the location of the active site, the substrate-binding groove and revealing the presence of a structurally important calcium ion. A zinc ion was found more loosely bound. Based on the structure, we propose a possible reaction mechanism. Future studies will be aimed at co-crystallizing CHAPK with substrate analogues and elucidating its role in the complete LysK protein. This, in turn, may lead to the design of site-directed mutants with altered activity or substrate specificity.The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under BioStruct-X grant agreement no. 283570 and was sponsored by grant BFU2011-24843 (MJvR) from the Spanish Ministry of Economy and Competitiveness, a Masters fellowship (MSG) and an FPU Ph.D. fellowship (CGD) from the Spanish Ministry of Education, Culture and Sports. We also acknowledge financial support from TSR-StrandIII:CRS/07/CR03 and FIRM:08RDCIT600 of the Irish Department of Agriculture

Crystal structure of the fibre head domain of bovine adenovirus 4, a ruminant atadenovirus

[Background] In adenoviruses, primary host cell recognition is generally performed by the head domains of their homo-trimeric fibre proteins. This first interaction is reversible. A secondary, irreversible interaction subsequently takes place via other adenovirus capsid proteins and leads to a productive infection. Although many fibre head structures are known for human mastadenoviruses, not many animal adenovirus fibre head structures have been determined, especially not from those belonging to adenovirus genera other than Mastadenovirus.[Methods] We constructed an expression vector for the fibre head domain from a ruminant atadenovirus, bovine adenovirus 4 (BAdV-4), consisting of amino acids 414–535, expressed the protein in Escherichia coli, purified it by metal affinity and cation exchange chromatography and crystallized it. The structure was solved using single isomorphous replacement plus anomalous dispersion of a mercury derivative and refined against native data that extended to 1.2 Å resolution.[Results] Like in other adenoviruses, the BAdV-4 fibre head monomer contains a beta-sandwich consisting of ABCJ and GHID sheets. The topology is identical to the fibre head of the other studied atadenovirus, snake adenovirus 1 (SnAdV-1), including the alpha-helix in the DG-loop, despite of them having a sequence identity of only 15 %. There are also differences which may have implications for ligand binding. Beta-strands G and H are longer and differences in several surface-loops and surface charge are observed.[Conclusions] Chimeric adenovirus fibres have been used to retarget adenovirus-based anti-cancer and gene therapy vectors. Ovine adenovirus 7 (OAdV-7), another ruminant atadenovirus, is intensively tested as a basis for such a vector. Here, we present the high-resolution atomic structure of the BAdV-4 fibre head domain, the second atadenovirus fibre head structure known and the first of an atadenovirus that infects a mammalian host. Future research should focus on the receptor-binding properties of these fibre head domains.The research leading to these results was sponsored by grant BFU2011-24843 (to MJvR, THN, MSG and AKS) from the Spanish Ministry of Economy and Competitiveness, a VAST-CSIC PhD fellowship to THN, a FEMS short-term Research Fellowship award to MZB, a RISAM fellowship to MSG, a La Caixa fellowship to AKS, and by grant OTKA NN107632 from the Hungarian Scientific Research Fund (to BH)

Self-Immolation of a Bacterial Dehydratase Enzyme by its Epoxide Product

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.Financial support from the Spanish Ministry of Economy and Competiveness (SAF2016‐75638‐R and BFU2017‐82207‐P), the Xunta de Galicia [ED431B 2018/04 and Centro singular de investigación de Galicia accreditation 2019‐2022 (ED431G 2019/03)] and the European Union (European Regional Development Fund—ERDF) is gratefully acknowledged. E.L. and M.M. thank the Xunta de Galicia for their respective postdoctoral fellowships. We are grateful to the ALBA synchrotron beamline XALOC (Barcelona, Spain) for the provision of beam time and to the Centro de Supercomputación de Galicia (CESGA) for use of the Finis Terrae computer

Structure and Function of Bacteriophages

Bacteriophages, or phages, are viruses with an exquisitely evolved structure to accomplish their goals. These goals are recognizing a suitable host bacterium, profiting from the host metabolism, and producing multiple progeny phages that are stable enough to survive until they find a new host bacterium to infect. Their genomes consist of single-stranded RNA, double-stranded RNA, single-stranded DNA, or double-stranded DNA, depending on phage type. They store their genome in highly symmetric protein capsids to protect it from degradation. Often these capsids are icosahedral, but helical and other shapes are also used. Tectiviridae and Corticoviridae have an internal lipid membrane, while Cystoviridae sport an outer membrane layer. Phages with tails, belonging to the Caudovirales order, are the most commonly encountered bacteriophages and have icosahedral or prolate capsids. In addition to the capsid, phages need a host cell recognition apparatus. The small icosahedral Leviviridae have a single minor capsid protein for this purpose. More complex phages dedicate multiple proteins to host cell recognition, and examples of this are the helical Inoviridae and the icosahedral Tectiviridae, Corticoviridae, and Cystoviridae. The Caudovirales have highly efficient tail protein complexes for DNA transfer. These tails are flexible (Siphoviridae), extensible (Podoviridae), or contractile (Myoviridae). Apart from elements designed for genome protection, host recognition, and genome transfer, more complicated phage particles may contain proteins for environmental sensing, binding to suitable matrices where host bacteria are likely to be encountered, and other functions