Four crystal structures of human LLT1, a ligand of human NKR-P1, in varied glycosylation and oligomerization states.

Human LLT1 is a C-type lectin-like ligand of NKR-P1 (CD161, gene KLRB1), a C-type lectin-like receptor of natural killer cells. Using X-ray diffraction, the first experimental structures of human LLT1 were determined. Four structures of LLT1 under various conditions were determined: monomeric, dimeric deglycosylated after the first N-acetylglucosamine unit in two forms and hexameric with homogeneous GlcNAc2Man5 glycosylation. The dimeric form follows the classical dimerization mode of human CD69. The monomeric form keeps the same fold with the exception of the position of an outer part of the long loop region. The hexamer of glycosylated LLT1 consists of three classical dimers. The hexameric packing may indicate a possible mode of interaction of C-type lectin-like proteins in the glycosylated form.

Mouse Clr-g, a ligand for NK cell activation receptor NKR-P1F: crystal structure and biophysical properties.

Interactions between C-type lectin-like NK cell receptors and their protein ligands form one of the key recognition mechanisms of the innate immune system that is involved in the elimination of cells that have been malignantly transformed, virally infected, or stressed by chemotherapy or other factors. We determined an x-ray structure for the extracellular domain of mouse C-type lectin related (Clr) protein g, a ligand for the activation receptor NKR-P1F. Clr-g forms dimers in the crystal structure resembling those of human CD69. This newly reported structure, together with the previously determined structure of mouse receptor NKR-P1A, allowed the modeling and calculations of electrostatic profiles for other closely related receptors and ligands. Despite the high similarity among Clr-g, Clr-b, and human CD69, these molecules have fundamentally different electrostatics, with distinct polarization of Clr-g. The electrostatic profile of NKR-P1F is complementary to that of Clr-g, which suggests a plausible interaction mechanism based on contacts between surface sites of opposite potential.

Plant multifunctional nuclease TBN1 with unexpected phospholipase activity: structural study and reaction-mechanism analysis.

Type I plant nucleases play an important role in apoptotic processes and cell senescence. Recently, they have also been indicated to be potent anticancer agents in in vivo studies. The first structure of tomato nuclease I (TBN1) has been determined, its oligomerization and activity profiles have been analyzed and its unexpected activity towards phospholipids has been discovered, and conclusions are drawn regarding its catalytic mechanism. The structure-solution process required X-ray diffraction data from two crystal forms. The first form was used for phase determination; the second form was used for model building and refinement. TBN1 is mainly α-helical and is stabilized by four disulfide bridges. Three observed oligosaccharides are crucial for its stability and solubility. The active site is localized at the bottom of the positively charged groove and contains a zinc cluster that is essential for enzymatic activity. An equilibrium between monomers, dimers and higher oligomers of TBN1 was observed in solution. Principles of the reaction mechanism of the phosphodiesterase activity are suggested, with central roles for the zinc cluster, the nucleobase-binding pocket (Phe-site) and Asp70, Arg73 and Asn167. Based on the distribution of surface residues, possible binding sites for dsDNA and other nucleic acids with secondary structure were identified. The phospholipase activity of TBN1, which is reported for the first time for a nuclease, significantly broadens the substrate promiscuity of the enzyme, and the resulting release of diacylglycerol, which is an important second messenger, can be related to the role of TBN1 in apoptosis.

Organophosphorus acid anhydrolase from Alteromonas macleodii: structural study and functional relationship to prolidases.

The bacterial enzyme organophosphorus acid anhydrolase (OPAA) is able to catalyze the hydrolysis of both proline dipeptides (Xaa-Pro) and several types of organophosphate (OP) compounds. The full three-dimensional structure of the manganese-dependent OPAA enzyme is presented for the first time. This enzyme, which was originally isolated from the marine bacterium Alteromonas macleodii, was prepared recombinantly in Escherichia coli. The crystal structure was determined at 1.8 Å resolution in space group C2, with unit-cell parameters a = 133.8, b = 49.2, c = 97.3 Å, ß = 125.0°. The enzyme forms dimers and their existence in solution was confirmed by dynamic light scattering and size-exclusion chromatography. The enzyme shares the pita-bread fold of its C-terminal domain with related prolidases. The binuclear manganese centre is located in the active site within the pita-bread domain. Moreover, an Ni(2+) ion from purification was localized according to anomalous signal. This study presents the full structure of this enzyme with complete surroundings of the active site and provides a critical analysis of its relationship to prolidases.

Structure of the human NK cell NKR-P1:LLT1 receptor:ligand complex reveals clustering in the immune synapse.

Signaling by the human C-type lectin-like receptor, natural killer (NK) cell inhibitory receptor NKR-P1, has a critical role in many immune-related diseases and cancer. C-type lectin-like receptors have weak affinities to their ligands; therefore, setting up a comprehensive model of NKR-P1-LLT1 interactions that considers the natural state of the receptor on the cell surface is necessary to understand its functions. Here we report the crystal structures of the NKR-P1 and NKR-P1:LLT1 complexes, which provides evidence that NKR-P1 forms homodimers in an unexpected arrangement to enable LLT1 binding in two modes, bridging two LLT1 molecules. These interaction clusters are suggestive of an inhibitory immune synapse. By observing the formation of these clusters in solution using SEC-SAXS analysis, by dSTORM super-resolution microscopy on the cell surface, and by following their role in receptor signaling with freshly isolated NK cells, we show that only the ligation of both LLT1 binding interfaces leads to effective NKR-P1 inhibitory signaling. In summary, our findings collectively support a model of NKR-P1:LLT1 clustering, which allows the interacting proteins to overcome weak ligand-receptor affinity and to trigger signal transduction upon cellular contact in the immune synapse.

Molecular architecture of mouse activating NKR-P1 receptors.

Receptors belonging to NKR-P1 family and their specific Clr ligands form an alternative missing self recognition system critical in immunity against tumors and viruses, elimination of tumor cells subjected to genotoxic stress, activation of T cell dependent immune response, and hypertension. The three-dimensional structure of the extracellular domain of the mouse natural killer (NK) cell receptor mNKR-P1Aex has been determined by X-ray diffraction. The core of the C-type lectin domain (CTLD) is homologous to the other CTLD receptors whereas one quarter of the domain forms an extended loop interacting tightly with a neighboring loop in the crystal. This domain swapping mechanism results in a compact interaction interface. A second dimerization interface resembles the known arrangement of other CTLD NK receptors. A functional dimeric form of the receptor is suggested, with the loop, evolutionarily conserved within this family, proposed to participate in interactions with ligands.

Structure of the H107R variant of the extracellular domain of mouse NKR-P1A at 2.3 Å resolution.

The structure of the H107R variant of the extracellular domain of the mouse natural killer cell receptor NKR-P1A has been determined by X-ray diffraction at 2.3 Å resolution from a merohedrally twinned crystal. Unlike the structure of the wild-type receptor in space group I4(1)22 with a single chain per asymmetric unit, the crystals of the variant belonged to space group I4(1) with a dimer in the asymmetric unit. Different degrees of merohedral twinning were detected in five data sets collected from different crystals. The mutation does not have a significant impact on the overall structure, but led to the binding of an additional phosphate ion at the interface of the molecules.

Structure of laccase from Streptomyces coelicolor after soaking with potassium hexacyanoferrate and at an improved resolution of 2.3†Å.

The paper reports the structure of the small laccase from Streptomyces coelicolor determined from a crystal soaked with potassium hexacyanoferrate [K4Fe(CN)6]. The decolorization of the natively blue crystal observed upon soaking indicates the reduction of the enzyme in the crystal. The ligand binds between laccase molecules and stabilizes the crystal. The increased diffraction limit of the diffraction data collected from this crystal enabled the refinement of the small laccase structure at 2.3 Šresolution, which is the highest resolution obtained to date.

Crystallization of recombinant bifunctional nuclease TBN1 from tomato.

The endonuclease TBN1 from Solanum lycopersicum (tomato) was expressed in Nicotiana benthamiana leaves and purified with suitable quality and in suitable quantities for crystallization experiments. Two crystal forms (orthorhombic and rhombohedral) were obtained and X-ray diffraction experiments were performed. The presence of natively bound Zn2+ ions was confirmed by X-ray fluorescence and by an absorption-edge scan. X-ray diffraction data were collected from the orthorhombic (resolution of 5.2 Å) and rhombohedral (best resolution of 3.2 Å) crystal forms. SAD, MAD and MR methods were applied for solution of the phase problem, with partial success. TBN1 contains three Zn2+ ions in a similar spatial arrangement to that observed in nuclease P1 from Penicillium citrinum.

Crystallographic fragment screening-based study of a novel FAD-dependent oxidoreductase from Chaetomium thermophilum.

The FAD-dependent oxidoreductase from Chaetomium thermophilum (CtFDO) is a novel thermostable glycoprotein from the glucose-methanol-choline (GMC) oxidoreductase superfamily. However, CtFDO shows no activity toward the typical substrates of the family and high-throughput screening with around 1000 compounds did not yield any strongly reacting substrate. Therefore, protein crystallography, including crystallographic fragment screening, with 42 fragments and 37 other compounds was used to describe the ligand-binding sites of CtFDO and to characterize the nature of its substrate. The structure of CtFDO reveals an unusually wide-open solvent-accessible active-site pocket with a unique His-Ser amino-acid pair putatively involved in enzyme catalysis. A series of six crystal structures of CtFDO complexes revealed five different subsites for the binding of aryl moieties inside the active-site pocket and conformational flexibility of the interacting amino acids when adapting to a particular ligand. The protein is capable of binding complex polyaromatic substrates of molecular weight greater than 500 Da.

Crystallographic fragment screening-based study of a novel FAD-dependent oxidoreductase from Chaetomium thermophilum. Corrigendum.

The synchrotron facilities used in collecting the data for the article by Svecová et al. [(2021), Acta Cryst. D77, 755-775] are acknowledged.

New insights into intra- and intermolecular interactions of immunoglobulins: crystal structure of mouse IgG2b-Fc at 2.1-A resolution.

The structure of the Fc fragment of monoclonal antibody IgG2b from hybridom M75 of Mus musculus has been determined by single crystal X-ray diffraction. This is the first report of the structure of the murine immunoglobulin isotype IgG2b. The structure refined at 2.1 A resolution provides more detailed structural information about native oligosaccharides than was previously available. High-quality Fourier maps provide a clear identification of alpha-l-fucose with partial occupancy in the first branch of the antennary oligosaccharides. A unique Fc:Fc interaction was observed at the C(H)2-C(H)3 interface.

Cystathionine gamma-lyase: Clinical, metabolic, genetic, and structural studies.

We report studies of six individuals with marked elevations of cystathionine in plasma and/or urine. Studies of CTH, the gene that encodes cystathionine gamma-lyase, revealed the presence among these individuals of either homozygous or compound heterozygous forms of a novel large deletion, p.Gly57_Gln196del, two novel missense mutations, c.589C>T (p.Arg197Cys) and c.932C>T (p.Thr311Ile), and one previously reported alteration, c.200C>T (p.Thr67Ile). Another novel missense mutation, c.185G>T (p.Arg62His), was found in heterozygous form in three mildly hypercystathioninemic members of a Taiwanese family. In one severely hypercystathioninemic individual no CTH mutation was found. Brief clinical histories of the cystathioninemic/cystathioninuric patients are presented. Most of the novel mutations were expressed and the CTH activities of the mutant proteins determined. The crystal structure of the human enzyme, hCTH, and the evidence available as to the effects of the mutations in question, as well as those of the previously reported p.Gln240Glu, on protein structure, enzymatic activity, and responsiveness to vitamin B(6) administration are discussed. Among healthy Czech controls, 9.3% were homozygous for CTH c.1208G>T (p.Ser403Ile), previously found homozygously in 7.5% of Canadians for whom plasma total homocysteine (tHcy) had been measured. Compared to wild-type homozygotes, among the 55 Czech c.1208G>T (p.Ser403Ile) homozygotes a greater level of plasma cystathionine was found only after methionine loading. Three of the four individuals homozygous or compound heterozygous for inactivating CTH mutations had mild plasma tHcy elevations, perhaps indicating a cause-and-effect relationship. The experience with the present patients provides no evidence that severe loss of CTH activity is accompanied by adverse clinical effects.

The high-resolution structure of the extracellular domain of human CD69 using a novel polymer.

The structure of the extracellular domain of human CD69 has been determined by single-crystal X-ray diffraction. The structure refined to 1.37 A resolution provides further details of the overall structure and the asymmetric interface between the monomers in the native dimer. The protein was crystallized using di[poly(ethylene glycol)] adipate, which also served as a cryoprotectant. This is the first report of a crystal structure determined using crystals grown with this polymer.

Crystallization of carbohydrate oxidase from Microdochium nivale.

Microdochium nivale carbohydrate oxidase was produced by heterologous recombinant expression in Aspergillus oryzae, purified and crystallized. The enzyme crystallizes with varying crystal morphologies depending on the crystallization conditions. Several different crystal forms were obtained using the hanging-drop vapour-diffusion method, two of which were used for diffraction measurements. Hexagon-shaped crystals (form I) diffracted to 2.66 A resolution, with unit-cell parameters a = b = 55.7, c = 610.4 A and apparent space group P6(2)22. Analysis of the data quality showed almost perfect twinning of the crystals. Attempts to solve the structure by molecular replacement did not give satisfactory results. Recently, clusters of rod-shaped crystals (form II) were grown in a solution containing PEG MME 550. These crystals belonged to the monoclinic system C2, with unit-cell parameters a = 132.9, b = 56.6, c = 86.5 A, beta = 95.7 degrees . Data sets were collected to a resolution of 2.4 A. The structure was solved by the molecular-replacement method. Model refinement is currently in progress.

Trp-His covalent adduct in bilirubin oxidase is crucial for effective bilirubin binding but has a minor role in electron transfer.

Unlike any protein studied so far, the active site of bilirubin oxidase from Myrothecium verrucaria contains a unique type of covalent link between tryptophan and histidine side chains. The role of this post-translational modification in substrate binding and oxidation is not sufficiently understood. Our structural and mutational studies provide evidence that this Trp396-His398 adduct modifies T1 copper coordination and is an important part of the substrate binding and oxidation site. The presence of the adduct is crucial for oxidation of substituted phenols and it substantially influences the rate of oxidation of bilirubin. Additionally, we bring the first structure of bilirubin oxidase in complex with one of its products, ferricyanide ion, interacting with the modified tryptophan side chain, Arg356 and the active site-forming loop 393-398. The results imply that structurally and chemically distinct types of substrates, including bilirubin, utilize the Trp-His adduct mainly for binding and to a smaller extent for electron transfer.

Crystallization and preliminary X-ray diffraction analysis of the small laccase from Streptomyces coelicolor.

The small bacterial laccase from the actinobacterium Streptomyces coelicolor which lacks the second of the three domains of the laccases structurally characterized to date was crystallized. This multi-copper phenol oxidase crystallizes in a primitive tetragonal lattice, with unit-cell parameters a = b = 179.8, c = 175.3 A. The crystals belong to either space group P4(1)2(1)2 or P4(3)2(1)2. The self-rotation function shows the presence of a noncrystallographic threefold axis in the structure. Phases will be determined from the anomalous signal of the natively present copper ions.

Cold-active beta-galactosidase from Arthrobacter sp. C2-2 forms compact 660 kDa hexamers: crystal structure at 1.9A resolution.

The X-ray structure of cold-active beta-galactosidase (isoenzyme C-2-2-1) from an Antarctic bacterium Arthrobacter sp. C2-2 was solved at 1.9A resolution. The enzyme forms 660 kDa hexamers with active sites opened to the central cavity of the hexamer and connected by eight channels with exterior solvent. To our best knowledge, this is the first cold-active beta-galactosidase with known structure and also the first known beta-galactosidase structure in the form of compact hexamers. The hexamer organization regulates access of substrates and ligands to six active sites and this unique packing, present also in solution, raises questions about its purpose and function. This enzyme belongs to glycosyl hydrolase family 2, similarly to Escherichia coli beta-galactosidase, forming tetramers necessary for its enzymatic function. However, we discovered significant differences between these two enzymes affecting the ability of tetramer/hexamer formation and complementation of the active site. This structure reveals new insights into the cold-adaptation mechanisms of enzymatic pathways of extremophiles.

HIV-1 protease mutations and inhibitor modifications monitored on a series of complexes. Structural basis for the effect of the A71V mutation on the active site.

Two new X-ray structures of an HIV-1 protease mutant (A71V, V82T, I84V) in complex with inhibitors SE and SQ, pseudotetrapeptide inhibitors with an acyclic S-hydroxyethylamine isostere, were determined. Comparison of eight structures exploring the binding of four similar inhibitors--SE, SQ (S-hydroxyethylamine isostere), OE (ethyleneamine), and QF34 (hydroxyethylene)--to wild-type and A71V/V82T/I84V HIV-1 protease elucidates the principles of altered interaction with changing conditions. The A71V mutation, which is distant from the active site, causes changes in the structure of the enzyme detectable by the means of X-ray structure analysis, and a route of propagation of the effect toward the active site is proposed.

Crystallization of nepenthesin I using a low-pH crystallization screen.

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 Šresolution, respectively. The crystals belonged to space group P212121, with unit-cell parameters a = 86.63, b = 95.90, c = 105.40 Å, α = ß = γ = 90° and a = 86.28, b = 97.22, c = 103.78 Å, α = ß = 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.