Cryogenic X-ray crystallographic studies of biomacromolecules at Turkish Light Source "Turkish DeLight".

X-ray crystallography is a robust and powerful structural biology technique that provides high-resolution atomic structures of biomacromolecules. Scientists use this technique to unravel mechanistic and structural details of biological macromolecules (e.g., proteins, nucleic acids, protein complexes, protein-nucleic acid complexes, or large biological compartments). Since its inception, single-crystal cryocrystallography has never been performed in Türkiye due to the lack of a single-crystal X-ray diffractometer. The X-ray diffraction facility recently established at the University of Health Sciences, Istanbul, Türkiye will enable Turkish and international researchers to easily perform high-resolution structural analysis of biomacromolecules from single crystals. Here, we describe the technical and practical outlook of a state-of-the-art home-source X-ray, using lysozyme as a model protein. The methods and practice described in this article can be applied to any biological sample for structural studies. Therefore, this article will be a valuable practical guide from sample preparation to data analysis.

Rapid and efficient ambient temperature X-ray crystal structure determination at Turkish Light Source.

High-resolution biomacromolecular structure determination is essential to better understand protein function and dynamics. Serial crystallography is an emerging structural biology technique which has fundamental limitations due to either sample volume requirements or immediate access to the competitive X-ray beamtime. Obtaining a high volume of well-diffracting, sufficient-size crystals while mitigating radiation damage remains a critical bottleneck of serial crystallography. As an alternative, we introduce the plate-reader module adapted for using a 72-well Terasaki plate for biomacromolecule structure determination at a convenience of a home X-ray source. We also present the first ambient temperature lysozyme structure determined at the Turkish light source (Turkish DeLight). The complete dataset was collected in 18.5 min with resolution extending to 2.39 Å and 100% completeness. Combined with our previous cryogenic structure (PDB ID: 7Y6A), the ambient temperature structure provides invaluable information about the structural dynamics of the lysozyme. Turkish DeLight provides robust and rapid ambient temperature biomacromolecular structure determination with limited radiation damage.

Oligomerization of A. thaliana Heterotrimeric G Protein Subunits AGB1 and AGG2 In Vitro.

Plant heterotrimeric G proteins are a major group of signaling molecules involved in regulation of critical processes including stress adaptation, seed size, grain quality and immune responses. Despite an abundance of in situ functional studies; purification of the individual subunits of the plant heterotrimer for biophysical and structural characterization and for studies on their interactions are lacking. In this study cloning of the genes encoding the ß subunit AGB1 of A. thaliana and its γ-subunits AGG1 and AGG2 using different E. coli expression vectors and screening of expression in several strains are reported. AGB1 could be expressed albeit at very low levels and in all cases it was accompanied by overexpression of E. coli chaperone proteins. AGG1 could only be detected in inclusion body fractions, whereas AGG2 was obtained in soluble fractions and was purified. Purified AGB1 and AGG2 subunits were shown to dimerize in vitro. Further characterization of AGG2 by small angle X-ray scattering measurements and by dynamic light scattering revealed that AGG2 formed homodimers with extended shape in solution. These results are also consistent with those from circular dichroism spectroscopy which yielded 39.4% helical and 50% random coil content for AGG2. This is the first study showing heterologous expression of a plant heterotrimeric G protein ß subunit individually and presenting its interaction with a plant γ-subunit in vitro. Results also show that the AGG2 subunit has a disordered structure, which would account for its role in diverse interactions for establishing selectivity in signal propagation.

Production of prebiotic 6-kestose using Zymomonas mobilis levansucrase in carob molasses and its effect on 5-HMF levels during storage.

Fructooligosaccharides have important potential use in the food industry due to their properties such as solubility in water, stability in acidity of fruit juices and during storage, low-calorie value and prebiotic effects. In this study, for the first time, Zymomonas mobilis levansucrase was used for in situ 6-kestose production in carob molasses. The produced kestose was stable during storage at 20 °C for 4 months. The product was evaluated for color, non-enzymatic browning index and titratable acidity during storage and the quality of the product was found comparable to that of control. Furthermore, the decreased amount of sucrose resulted in the prevention of 5-hydroxymethylfurfural (5-HMF) formation during storage. As a result, carob molasses was converted into a high-quality prebiotic product with decreased sucrose content and reduced 5-HMF quantities, and a new method was developed to prevent 5-HMF formation in fruit juices and molasses.

A putative Toll/interleukin-1 receptor domain protein from Helicobacter pylori is dimeric in solution and interacts with human Toll-like receptor adaptor myeloid differentiation primary response 88.

Helicobacter pylori, an important human pathogen, is capable of causing persistent infection with minimal immune response. The first line of defense during H. pylori infection is through gastric epithelial cells that present TLR, A family of bacterial proteins that share homology with the Toll/IL-1 receptor (TIR) domain were identified. Bacterial TIR proteins (BTP) mimic human TIR domain proteins and act on myeloid differentiation primary response gene 88 (MyD88) signaling pathways to suppress TLR signaling. H. pylori may also produce a similar protein. A putative H. pylori BTP was found based on sequence homology. The corresponding gene hp1437 was inserted into an expression vector in fusion with an N-terminal cleavable 6his-tag. The recombinant protein, 6his-HP1437, was purified using nickel affinity chromatography with a yield of 8 mg/L culture. Oligomerization of HP1437 was investigated by size-exclusion chromatography. It was found that HP1437 forms dimers in solution similar to other BTPs. Furthermore, glutathione S-transferase pull down assays identified an interaction between HP1437 and human TIR domain adaptor MyD88. These findings suggest that HP1437 has the characteristic features of BTPs and may play a direct role in reducing immune response against H. pylori by binding to MyD88 and pave the way for an in-depth characterization of this putative novel H. pylori virulence factor.

The Brucella TIR domain containing proteins BtpA and BtpB have a structural WxxxE motif important for protection against microtubule depolymerisation.

BACKGROUND: The TIR domain-containing proteins BtpA/Btp1/TcpB and BtpB are translocated into host cells by the facultative intracellular bacterial pathogen Brucella. Here, they interfere with Toll like receptor signalling to temper the host inflammatory response. BtpA has also been found to modulate microtubule dynamics. In both proteins we identified a WxxxE motif, previously shown to be an essential structural component in a family of bacterial type III secretion system effectors that modulate host actin dynamics by functioning as guanine nucleotide exchange factors of host GTPases. We analysed a role for the WxxxE motif in association of BtpA and BtpB with the cytoskeleton. RESULTS: Unlike BtpA, ectopically expressed BtpB did not show a tubular localisation, but was found ubiquitously in the cytoplasm and the nucleus, and often appeared in discrete punctae in HeLa cells. BtpB was able to protect microtubules from drug-induced destabilisation similar to BtpA. The WxxxE motif was important for the ability of BtpA and BtpB to protect microtubules against destabilising drugs. Surprisingly, ectopic expression of BtpA, although not BtpB, in HeLa cells induced the formation of filopodia. This process was invariably dependent of the WxxxE motif. Our recent resolution of the crystal structure of the BtpA TIR domain reveals that the motif positions a glycine residue that has previously been shown to be essential for interaction of BtpA with microtubules. CONCLUSIONS: Our results suggest a structural role for the WxxxE motif in the association of BtpA and BtpB with microtubules, as with the WxxxE GEF family proteins where the motif positions an adjacent catalytic loop important for interaction with specific Rho GTPases. In addition, the ability of ectopically expressed BtpA to induce filopodia in a WxxxE-dependent manner suggests a novel property for BtpA. A conserved WxxxE motif is found in most bacterial and several eukaryotic TIR domain proteins. Despite the similarity between ectopically expressed BtpA and WxxxE GEFs to modulate host actin dynamics, our results suggest that BtpA is not part of this WxxxE GEF family. The WxxxE motif may therefore be a more common structural motif than thus far described. BtpA may provide clues to cross-talk between the TLR and GTPase signalling pathways.

Structure of the Toll/interleukin 1 receptor (TIR) domain of the immunosuppressive Brucella effector BtpA/Btp1/TcpB.

BtpA/Btp1/TcpB is a virulence factor produced by Brucella species that possesses a Toll interleukin-1 receptor (TIR) domain. Once delivered into the host cell, BtpA interacts with MyD88 to interfere with TLR signalling and modulates microtubule dynamics. Here the crystal structure of the BtpA TIR domain at 3.15 Å is presented. The structure shows a dimeric arrangement of a canonical TIR domain, similar to the Paracoccus denitrificans Tir protein but secured by a unique long N-terminal α-tail that packs against the TIR:TIR dimer. Structure-based mutations and multi-angle light scattering experiments characterized the BtpA dimer conformation in solution. The structure of BtpA will help with studies to understand the mechanisms involved in its interactions with MyD88 and with microtubules.

Structural insights into Helicobacter pylori oncoprotein CagA interaction with ß1 integrin.

Infection with the gastric pathogen Helicobacter pylori is a risk factor for the development of gastric cancer. Pathogenic strains of H. pylori carry a type IV secretion system (T4SS) responsible for the injection of the oncoprotein CagA into host cells. H. pylori and its cag-T4SS exploit α5ß1 integrin as a receptor for CagA translocation. Injected CagA localizes to the inner leaflet of the host cell membrane, where it hijacks host cell signaling and induces cytoskeleton reorganization. Here we describe the crystal structure of the N-terminal ~100-kDa subdomain of CagA at 3.6 Å that unveils a unique combination of folds. The core domain of the protein consists of an extended single-layer ß-sheet stabilized by two independent helical subdomains. The core is followed by a long helix that forms a four-helix helical bundle with the C-terminal domain. Mapping of conserved regions in a set of CagA sequences identified four conserved surface-exposed patches (CSP1-4), which represent putative hot-spots for protein-protein interactions. The proximal part of the single-layer ß-sheet, covering CSP4, is involved in specific binding of CagA to the ß1 integrin, as determined by yeast two-hybrid and in vivo competition assays in H. pylori cell-culture infection studies. These data provide a structural basis for the first step of CagA internalization into host cells and suggest that CagA uses a previously undescribed mechanism to bind ß1 integrin to mediate its own translocation.