Stimulator of interferon genes (STING) binds cyclic dinucleotides (CDNs), which induce a large conformational change of the protein. The structural basis of activation of STING by CDNs is rather well understood. Unliganded STING forms an open dimer that undergoes a large conformational change (∼10 Å) to a closed conformation upon the binding of a CDN molecule. This event activates downstream effectors of STING and subsequently leads to activation of the type 1 interferon response. However, a previously solved structure of STING with 3',3'-c-di-GMP shows Mg atoms mediating the interaction of STING with this CDN. Here, it is shown that no Mg atoms are needed for this interaction; in fact, magnesium can in some cases obstruct the binding of a CDN to STING.
Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.
- MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- genetické vektory chemie metabolismus MeSH
- halogenované uhlovodíky chemie metabolismus MeSH
- hydrolasy chemie genetika metabolismus MeSH
- interakční proteinové domény a motivy MeSH
- klonování DNA MeSH
- konformace proteinů, alfa-helix MeSH
- konformace proteinů, beta-řetězec MeSH
- krystalografie rentgenová MeSH
- nízká teplota MeSH
- Psychrobacter chemie enzymologie MeSH
- rekombinantní fúzní proteiny chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- simulace molekulového dockingu MeSH
- strukturní homologie proteinů MeSH
- substrátová specifita MeSH
- termodynamika MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
Human aldo-keto reductase 1C3 (AKR1C3) stereospecifically reduces steroids and prostaglandins and is involved in the biotransformation of xenobiotics. Its role in various cancers makes it a potential therapeutic target for the development of inhibitors. Recombinant AKR1C3 with a thrombin-cleavable N-terminal His6 tag was expressed from a pET-28(+) vector for structural studies of enzyme-inhibitor complexes. A modified in situ proteolysis approach was applied to specifically remove the His tag by thrombin cleavage during crystallization screening trials. This improved the morphology and diffraction quality of the crystals and allowed the acquisition of high-resolution diffraction data and structure solution. This approach may be generally applicable to other proteins expressed using the pET-28(+) vector.
- MeSH
- difrakce rentgenového záření metody MeSH
- histidin * genetika MeSH
- krystalizace metody MeSH
- krystalografie rentgenová metody MeSH
- lidé MeSH
- protein AKR1C3 chemie genetika metabolismus MeSH
- proteolýza MeSH
- sekvence aminokyselin MeSH
- thrombin metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
14-3-3 proteins bind phosphorylated binding partners to regulate several of their properties, including enzymatic activity, stability and subcellular localization. Here, two crystal structures are presented: the crystal structures of the 14-3-3 protein (also known as Bmh1) from the yeast Lachancea thermotolerans in the unliganded form and bound to a phosphopeptide derived from human PI4KB (phosphatidylinositol 4-kinase B). The structures demonstrate the high evolutionary conservation of ligand recognition by 14-3-3 proteins. The structural analysis suggests that ligand recognition by 14-3-3 proteins evolved very early in the evolution of eukaryotes and remained conserved, underlying the importance of 14-3-3 proteins in physiology.
- MeSH
- 1-fosfatidylinositol-4-kinasa chemie genetika metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- exprese genu MeSH
- fosfoproteiny chemie genetika metabolismus MeSH
- fungální proteiny chemie genetika metabolismus MeSH
- klonování DNA MeSH
- konformace proteinů, alfa-helix MeSH
- konzervovaná sekvence MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- ligandy MeSH
- molekulární evoluce MeSH
- molekulární modely MeSH
- plazmidy chemie metabolismus MeSH
- protein - isoformy chemie genetika metabolismus MeSH
- proteiny 14-3-3 chemie genetika metabolismus MeSH
- rekombinantní proteiny chemie genetika metabolismus MeSH
- Saccharomycetales chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- strukturní homologie proteinů MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The HsdR subunit of the type I restriction-modification system EcoR124I is responsible for the translocation as well as the restriction activity of the whole complex consisting of the HsdR, HsdM and HsdS subunits, and while crystal structures are available for the wild type and several mutants, the C-terminal domain comprising approximately 150 residues was not resolved in any of these structures. Here, three fusion constructs with the GFP variant pHluorin developed to overexpress, purify and crystallize the C-terminal domain of HsdR are reported. The shortest of the three encompassed HsdR residues 887-1038 and yielded crystals that belonged to the orthorhombic space group C2221, with unit-cell parameters a = 83.42, b = 176.58, c = 126.03 Å, α = β = γ = 90.00° and two molecules in the asymmetric unit (VM = 2.55 Å(3) Da(-1), solvent content 50.47%). X-ray diffraction data were collected to a resolution of 2.45 Å.
- MeSH
- difrakce rentgenového záření MeSH
- Escherichia coli chemie enzymologie genetika MeSH
- exprese genu MeSH
- klonování DNA MeSH
- krystalizace MeSH
- krystalografie rentgenová MeSH
- plazmidy chemie metabolismus MeSH
- podjednotky proteinů chemie genetika metabolismus MeSH
- proteiny z Escherichia coli chemie genetika metabolismus MeSH
- rekombinantní fúzní proteiny chemie genetika metabolismus MeSH
- restrikční endonukleasy typu I chemie genetika metabolismus MeSH
- sekvence aminokyselin MeSH
- zelené fluorescenční proteiny chemie genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
Hemihedral twinning is a crystal-growth anomaly in which a specimen is composed of two crystal domains that coincide with each other in three dimensions. However, the orientations of the crystal lattices in the two domains differ in a specific way. In diffraction data collected from hemihedrally twinned crystals, each observed intensity contains contributions from both of the domains. With perfect hemihedral twinning, the two domains have the same volumes and the observed intensities do not contain sufficient information to detwin the data. Here, the use of molecular replacement and of noncrystallographic symmetry (NCS) averaging to detwin a 2.1 Å resolution data set for Aichi virus 1 affected by perfect hemihedral twinning is described. The NCS averaging enabled the correction of errors in the detwinning introduced by the differences between the molecular-replacement model and the crystallized structure. The procedure permitted the structure to be determined from a molecular-replacement model that had 16% sequence identity and a 1.6 Å r.m.s.d. for C(α) atoms in comparison to the crystallized structure. The same approach could be used to solve other data sets affected by perfect hemihedral twinning from crystals with NCS.
Nepenthesins are aspartic proteases secreted by carnivorous pitcher plants of the genus Nepenthes. They significantly differ in sequence from other plant aspartic proteases. This difference, which provides more cysteine residues in the structure of nepenthesins, may contribute to their unique stability profile. Recombinantly produced nepenthesin 1 (rNep1) from N. gracilis in complex with pepstatin A was crystallized under two different crystallization conditions using a newly formulated low-pH crystallization screen. The diffraction data were processed to 2.9 and 2.8 Å resolution, respectively. The crystals belonged to space group P212121, with unit-cell parameters a = 86.63, b = 95.90, c = 105.40 Å, α = β = γ = 90° and a = 86.28, b = 97.22, c = 103.78 Å, α = β = γ = 90°, respectively. Matthews coefficient and solvent-content calculations suggest the presence of two molecules of rNep1 in the asymmetric unit. Here, the details of the crystallization experiment and analysis of the X-ray data are reported.
The glyceraldehyde dehydrogenase from Thermoplasma acidophilum (TaAlDH) is a microbial enzyme that catalyzes the oxidation of D-glyceraldehyde to D-glycerate in the artificial enzyme cascade designed for the conversion of glucose to the organic solvents isobutanol and ethanol. Various mutants of TaAlDH were constructed by a random approach followed by site-directed and saturation mutagenesis in order to improve the properties of the enzyme that are essential for its functioning within the cascade. Two enzyme variants, wild-type TaAlDH (TaAlDHwt) and an F34M+S405N variant (TaAlDH F34M+S405N), were successfully crystallized. Crystals of TaAlDHwt belonged to the monoclinic space group P1211 with eight molecules per asymmetric unit and diffracted to a resolution of 1.95 Å. TaAlDH F34M+S405N crystallized in two different space groups: triclinic P1 with 16 molecules per asymmetric unit and monoclinic C121 with four molecules per asymmetric unit. These crystals diffracted to resolutions of 2.14 and 2.10 Å for the P1 and C121 crystals, respectively.
Tomato multifunctional nuclease TBN1 belongs to the type I nuclease family, which plays an important role in apoptotic processes and cell senescence in plants. The newly solved structure of the N211D mutant is reported. Although the main crystal-packing motif (the formation of superhelices) is conserved, the details differ among the known structures. A phosphate ion was localized in the active site of the enzyme. The binding of the surface loop to the active centre is stabilized by the phosphate ion, which correlates with the observed aggregation of TBN1 in phosphate buffer. The conserved binding of the surface loop to the active centre suggests biological relevance of the contact in a regulatory function or in the formation of oligomers.
- MeSH
- endodeoxyribonukleasy chemie genetika metabolismus MeSH
- fosfáty metabolismus MeSH
- krystalizace MeSH
- molekulární sekvence - údaje MeSH
- multienzymové komplexy chemie genetika metabolismus MeSH
- rostlinné proteiny chemie genetika metabolismus MeSH
- sekundární struktura proteinů MeSH
- sekvence aminokyselin MeSH
- Solanum lycopersicum enzymologie genetika MeSH
- vazebná místa fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- internacionalita MeSH
- krystalizace metody MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- makromolekulární látky chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- úvodní články MeSH
