The first Federation of European Biochemical Societies Advanced Course on macromolecular crystallization was launched in the Czech Republic in October 2004. Over the past two decades, the course has developed into a distinguished event, attracting students, early career postdoctoral researchers and lecturers. The course topics include protein purification, characterization and crystallization, covering the latest advances in the field of structural biology. The many hands-on practical exercises enable a close interaction between students and teachers and offer the opportunity for students to crystallize their own proteins. The course has a broad and lasting impact on the scientific community as participants return to their home laboratories and act as nuclei by communicating and implementing their newly acquired knowledge and skills.

The T3SS is utilized by many Gram-negative bacteria to deliver effector proteins from bacterial cytosol directly into infected host cell cytoplasm in a regulated and targeted manner. Pathogenic bordetellae use the T3SS to inject the BteA and BopN proteins into infected cells and upregulate the production of the anti-inflammatory cytokine interleukin-10 (IL-10) to evade host immunity.

Iripin-4, one of the many salivary serpins from Ixodes ricinus ticks with an as-yet unexplained function, crystallized in two different structural conformations, namely the native partially relaxed state and the cleaved serpin. The native structure was solved at a resolution of 2.3 Å and the structure of the cleaved conformation was solved at 2.0 Å resolution. Furthermore, structural changes were observed when the reactive-centre loop transitioned from the native conformation to the cleaved conformation. In addition to this finding, it was confirmed that Glu341 represents a primary substrate-recognition site for the inhibitory mechanism. The presence of glutamate instead of the typical arginine in the P1 recognition site of all structurally characterized I. ricinus serpins (PDB entries 7b2t, 7pmu and 7ahp), except for the tyrosine in the P1 site of Iripin-2 (formerly IRS-2; PDB entry 3nda), would explain the absence of inhibition of the tested proteases that cleave their substrate after arginine. Further research on Iripin-4 should focus on functional analysis of this interesting serpin.

Nowadays, X-ray crystallography is one of the most popular structural biology methods. Successful crystallization depends not only on the quality of the protein sample, precipitant composition, pH or other biophysical and biochemical parameters, but also largely on the use of crystallization technique. Some proteins are difficult to be crystallized using basic crystallization methods; therefore, several advanced methods for macromolecular crystallization have been developed. This chapter briefly reviews the most promising advanced crystallization techniques and strategies as one of the efficient tools for crystallization of macromolecules. Crystallization in capillaries, gels, microfluidic chips, electric and magnetic fields as well as crystallization under microgravity condition and crystallization in living cells are briefly described.

Haloalkane dehalogenases (EC 3.8.1.5) are microbial enzymes that catalyse the hydrolytic conversion of halogenated compounds, resulting in a halide ion, a proton and an alcohol. These enzymes are used in industrial biocatalysis, bioremediation and biosensing of environmental pollutants or for molecular tagging in cell biology. The novel haloalkane dehalogenase DpaA described here was isolated from the psychrophilic and halophilic bacterium Paraglaciecola agarilytica NO2, which was found in marine sediment collected from the East Sea near Korea. Gel-filtration experiments and size-exclusion chromatography provided information about the dimeric composition of the enzyme in solution. The DpaA enzyme was crystallized using the sitting-drop vapour-diffusion method, yielding rod-like crystals that diffracted X-rays to 2.0 Å resolution. Diffraction data analysis revealed a case of merohedral twinning, and subsequent structure modelling and refinement resulted in a tetrameric model of DpaA, highlighting an uncommon multimeric nature for a protein belonging to haloalkane dehalogenase subfamily I.

Iripin-5 is the main Ixodes ricinus salivary serpin, which acts as a modulator of host defence mechanisms by impairing neutrophil migration, suppressing nitric oxide production by macrophages and altering complement functions. Iripin-5 influences host immunity and shows high expression in the salivary glands. Here, the crystal structure of Iripin-5 in the most thermodynamically stable state of serpins is described. In the reactive-centre loop, the main substrate-recognition site of Iripin-5 is likely to be represented by Arg342, which implies the targeting of trypsin-like proteases. Furthermore, a computational structural analysis of selected Iripin-5-protease complexes together with interface analysis revealed the most probable residues of Iripin-5 involved in complex formation.

Antimicrobial peptides form part of the innate immune response and play a vital role in host defense against pathogens. Here we report a new antimicrobial peptide belonging to the cathelicidin family, cathelicidin-MH (cath-MH), from the skin of Microhyla heymonsivogt frog. Cath-MH has a single α-helical structure in membrane-mimetic environments and is antimicrobial against fungi and bacteria, especially Gram-negative bacteria. In contrast to other cathelicidins, cath-MH suppresses coagulation by affecting the enzymatic activities of tissue plasminogen activator, plasmin, β-tryptase, elastase, thrombin, and chymase. Cath-MH protects against lipopolysaccharide (LPS)- and cecal ligation and puncture-induced sepsis, effectively ameliorating multiorgan pathology and inflammatory cytokine through its antimicrobial, LPS-neutralizing, coagulation suppressing effects as well as suppression of MAPK signaling. Taken together, these data suggest that cath-MH is an attractive candidate therapeutic agent for the treatment of septic shock.

Tick saliva is a rich source of pharmacologically and immunologically active molecules. These salivary components are indispensable for successful blood feeding on vertebrate hosts and are believed to facilitate the transmission of tick-borne pathogens. Here we present the functional and structural characterization of Iripin-3, a protein expressed in the salivary glands of the tick Ixodes ricinus, a European vector of tick-borne encephalitis and Lyme disease. Belonging to the serpin superfamily of protease inhibitors, Iripin-3 strongly inhibited the proteolytic activity of serine proteases kallikrein and matriptase. In an in vitro setup, Iripin-3 was capable of modulating the adaptive immune response as evidenced by reduced survival of mouse splenocytes, impaired proliferation of CD4+ T lymphocytes, suppression of the T helper type 1 immune response, and induction of regulatory T cell differentiation. Apart from altering acquired immunity, Iripin-3 also inhibited the extrinsic blood coagulation pathway and reduced the production of pro-inflammatory cytokine interleukin-6 by lipopolysaccharide-stimulated bone marrow-derived macrophages. In addition to its functional characterization, we present the crystal structure of cleaved Iripin-3 at 1.95 Å resolution. Iripin-3 proved to be a pluripotent salivary serpin with immunomodulatory and anti-hemostatic properties that could facilitate tick feeding via the suppression of host anti-tick defenses. Physiological relevance of Iripin-3 activities observed in vitro needs to be supported by appropriate in vivo experiments.

The antibiotic resistance incidence is alarmingly increasing in both human and veterinary medicine and is one of the major concerns of contemporary healthcare.Because most of the currently used antibiotics have natural analogs with similar native structures, AMRassociated genes are widely present in bacteria in the environment, and they can be easily distributed to clinically important strains through horizontal gene transfer.Such bacteria pose a threat primarily in hospitals, where infections of immunocompromised patients often end fatally.A small group of bacteria causing nosocomial infections are present in both the developed and developing world; it is called ESKAPE in abbreviation and is comprised of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and other Enterobacteriaceae species.These pathogens represent the most significant threat among the so-called superbugs, which can rapidly acquire resistance to several classes of antibiotics and can cause a variety of nosocomial infections, mostly in the respiratory or urinary tract, but bloodstream, wound, and skin infections are also frequent.Overall, nosocomial infections result in increased mortality and morbidity rate in the affected patients.For endolysins targeting Gram-negative bacteria the specific activity against a broad host range is a known phenomenon, however, the molecular mechanisms specifying their broad spectrum of action are obscure.There is still no global understanding, whether bacterial cell lysis with endolysins is determined by the action of the enzymatic activity of the proteins or by additional functional domains containing in their structure, or the action of endolysins against Gram-negative microorganisms is determined by the joint interaction of several distinct domains.Here we propose to investigate in details the structural aspects of LysSi3 endolysin mechanism of action.The LysSi3 is a peptidoglycan hydrolyzing, lysozyme-like enzyme with predicted muramidase activity (GH24 family) and broad bactericidal activity against ESKAPE pathogens.

The activity of enzymes with active sites buried inside their protein core highly depends on the efficient transport of substrates and products between the active site and the bulk solvent. The engineering of access tunnels in order to increase or decrease catalytic activity and specificity in a rational way is a challenging task. Here, we describe a combined experimental and computational approach to characterize the structural basis of altered activity in the haloalkane dehalogenase LinB D147C+L177C variant. While the overall protein fold is similar to the wild type enzyme and the other LinB variants, the access tunnels have been altered by introduced cysteines that were expected to form a disulfide bond. Surprisingly, the mutations have allowed several conformations of the amino acid chain in their vicinity, interfering with the structural analysis of the mutant by X-ray crystallography. The duration required for the growing of protein crystals changed from days to 1.5 years by introducing the substitutions. The haloalkane dehalogenase LinB D147C+L177C variant crystal structure was solved to 1.15 Å resolution, characterized and deposited to Protein Data Bank under PDB ID 6s06.

Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biology. The method of ancestral sequence reconstruction leads to prediction of sequences of ancestral enzymes allowing their experimental characterization. Based on the sequences of modern haloalkane dehalogenases from the subfamily II, the most common ancestor of thoroughly characterized enzymes LinB from Sphingobium japonicum UT26 and DmbA from Mycobacterium bovis 5033/66 was in silico predicted, recombinantly produced and structurally characterized. The ancestral enzyme AncLinB-DmbA was crystallized using the sitting-drop vapor-diffusion method, yielding rod-like crystals that diffracted X-rays to 1.5 Å resolution. Structural comparison of AncLinB-DmbA with their closely related descendants LinB and DmbA revealed some differences in overall structure and tunnel architecture. Newly prepared AncLinB-DmbA has the highest active site cavity volume and the biggest entrance radius on the main tunnel in comparison to descendant enzymes. Ancestral sequence reconstruction is a powerful technique to study molecular evolution and design robust proteins for enzyme technologies.

Ionic liquids attracted interest as green alternatives to replace conventional organic solvents in protein stability studies. They can play an important role in the stabilization of enzymes such as haloalkane dehalogenases that are used for biodegradation of warfare agents and halogenated environmental pollutants. Three-dimensional crystals of haloalkane dehalogenase variant DhaA80 (T148L+G171Q+A172V+C176F) from Rhodococcus rhodochrous NCIMB 13064 were grown and soaked with the solutions of 2-hydroxyethylammonium acetate and 1-butyl-3-methylimidazolium methyl sulfate. The objective was to study the structural basis of the interactions between the ionic liquids and the protein. The diffraction data were collected for the 1.25 Å resolution for 2-hydroxyethylammonium acetate and 1.75 Å resolution for 1-butyl-3-methylimidazolium methyl sulfate. The structures were used for molecular dynamics simulations to study the interactions of DhaA80 with the ionic liquids. The findings provide coherent evidence that ionic liquids strengthen both the secondary and tertiary protein structure due to extensive hydrogen bond interactions.