Ute Krengel

Function of the CysD domain of the gel-forming MUC2 mucin.

The colonic human MUC2 mucin forms a polymeric gel by covalent disulfide bonds in its N- and C-termini. The middle part of MUC2 is largely composed of two highly O-glycosylated mucin domains that are interrupted by a CysD domain of unknown function. We studied its function as recombinant proteins fused to a removable immunoglobulin Fc domain. Analysis of affinity-purified fusion proteins by native gel electrophoresis and gel filtration showed that they formed oligomeric complexes. Analysis of the individual isolated CysD parts showed that they formed dimers both when flanked by two MUC2 tandem repeats and without these. Cleavages of the two non-reduced CysD fusion proteins and analysis by MS revealed the localization of all five CysD disulfide bonds and that the predicted C-mannosylated site was not glycosylated. All disulfide bonds were within individual peptides showing that the domain was stabilized by intramolecular disulfide bonds and that CysD dimers were of non-covalent nature. These observations suggest that CysD domains act as non-covalent cross-links in the MUC2 gel, thereby determining the pore sizes of the mucus.

High-resolution Crystal Structures of Erythrina cristagalli Lectin in Complex with Lactose and 2′-α-l-Fucosyllactose and Correlation with Thermodynamic Binding Data

The primary sequence of Erythrina cristagalli lectin (ECL) was mapped by mass spectrometry, and the crystal structures of the lectin in complex with lactose and 2-alpha-L-fucosyllactose were determined at 1.6A and 1.7A resolution, respectively. The two complexes were compared with the crystal structure of the closely related Erythrina corallodendron lectin (ECorL) in complex with lactose, with the crystal structure of the Ulex europaeus lectin II in complex with 2-alpha-L-fucosyllactose, and with two modeled complexes of ECorL with 2-alpha-L-fucosyl-N-acetyllactosamine. The molecular models are very similar to the crystal structure of ECL in complex with 2-alpha-L-fucosyllactose with respect to the overall mode of binding, with the L-fucose fitting snugly into the cavity surrounded by Tyr106, Tyr108, Trp135 and Pro134 adjoining the primary combining site of the lectin. Marked differences were however noted between the models and the experimental structure in the network of hydrogen bonds and hydrophobic interactions holding the L-fucose in the combining site of the lectin, pointing to limitations of the modeling approach. In addition to the structural characterization of the ECL complexes, an effort was undertaken to correlate the structural data with thermodynamic data obtained from microcalorimetry, revealing the importance of the water network in the lectin combining site for carbohydrate binding.

Glucose kinase of Streptomyces coelicolor A3(2): large-scale purification and biochemical analysis

Glucose kinase of Streptomyces coelicolor A3(2) is essential for glucose utilisation and is required for carbon catabolite repression (CCR) exerted through glucose and other carbon sources. The protein belongs to the ROK-family, which comprises bacterial sugar kinases and regulators. To better understand glucose kinase function, we have monitored the cellular activity and demonstrated that the choice of carbon sources did not significantly change the synthesis and activity of the enzyme. The DNA sequence of the Streptomyces lividans glucose kinase gene glkA was determined. The predicted gene product of 317 amino acids was found to be identical to S. coelicolor glucose kinase, suggesting a similar role for this protein in both organisms. A procedure was developed to produce pure histidine-tagged glucose kinase with a yield of approximately 10 mg/l culture. The protein was stable for several weeks and was used to raise polyclonal antibodies. Purified glucose kinase was used to explore protein-protein interaction by surface plasmon resonance. The experiments revealed the existence of a binding activity present in S. coelicolor cell extracts. This indicated that glucose kinase may interact with (an)other factor(s), most likely of protein nature. A possible cross-talk with proteins of the phosphotransferase system, which are involved in carbon catabolite repression in other bacteria, was investigated.

Structure and molecular interactions of a unique antitumor antibody specific for N-glycolyl GM3

N-Glycolyl GM3 ganglioside is an attractive target antigen for cancer immunotherapy, because this epitope is a molecular marker of certain tumor cells and not expressed in normal human tissues. The murine monoclonal antibody 14F7 specifically recognizes N-glycolyl GM3 and shows no cross-reactivity with the abundant N-acetyl GM3 ganglioside, a close structural homologue of N-glycolyl GM3. Here, we report the crystal structure of the 14F7 Fab fragment at 2.5 Å resolution and its molecular model with the saccharide moiety of N-glycolyl GM3, NeuGcα3Galβ4Glcβ. Fab 14F7 contains a very long CDR H3 loop, which divides the antigen-binding site of this antibody into two subsites. In the docking model, the saccharide ligand is bound to one of these subsites, formed solely by heavy chain residues. The discriminative feature of N-glycolyl GM3 versus N-acetyl GM3, its hydroxymethyl group, is positioned in a hydrophilic cavity, forming hydrogen bonds with the carboxyl group of Asp H52, the indole NH of Trp H33 and the hydroxyl group of Tyr H50. For the hydrophobic methyl group of N-acetyl GM3, this environment would not be favorable, explaining why the antibody specifically recognizes N-glycolyl GM3, but not N-acetyl GM3. Mutation of Asp H52 to hydrophobic residues of similar size completely abolished binding. Our model of the antibodycarbohydrate complex is consistent with binding data for several tested glycolipids as well as for a variety of 14F7 mutants with replaced VL domains.

Crystal Structures Exploring the Origins of the Broader Specificity of Escherichia coli Heat-Labile Enterotoxin Compared to Cholera Toxin

Cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) are structurally and functionally related and share the same primary receptor, the GM1 ganglioside. Despite their extensive similarities, these two toxins exhibit distinct ligand specificities, with LT being more promiscuous than CT. Here, we have attempted to rationalize the broader binding specificity of LT and the subtle differences between the binding characteristics of LTs from human and porcine origins (mediated by their B subunit pentamers, hLTB and pLTB, respectively). The analysis is based on two crystal structures of pLTB in complexes with the pentasaccharide of its primary ligand, GM1, and with neolactotetraose, the carbohydrate determinant of a typical secondary ligand of LTs, respectively. Important molecular determinants underlying the different binding specificities of LTB and CTB are found to be contributed by Ser95, Tyr18 and Thr4 (or Ser4 of hLTB), which together prestabilize the binding site by positioning Lys91, Glu51 and the adjacent loop region (50-61) containing Ile58 for ligand binding. Glu7 and Ala1 may also play an important role. Many of these residues are closely connected with a recently identified second binding site, and there appears to be cross-talk between the two sites. Binding to N-acetyllactosamine-terminated receptors is further augmented by Arg13 (present in pLT and some hLT variants), as previously predicted.

Preliminary X-ray crystallographic analysis of the secreted chorismate mutase from Mycobacterium tuberculosis: a tricky crystallization problem solved

A method is presented that allowed the diffraction limit of crystals of the secreted chorismate mutase from M. tuberculosis to be improved from approximately 3.5 to 1.3u2005Å. To obtain large well diffracting crystals, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5–15u2005min.

Crystallization and preliminary X-ray crystallographic studies of a lectin from the mushroom Marasmius oreades.

The Marasmius oreades agglutinin (MOA) recognizes blood group B oligosaccharides. This mushroom lectin belongs to the ricin superfamily and is currently the only lectin known with exclusive specificity for Galalpha1,3Gal-structures, as occur in the subterminally fucosylated blood group B epitope Galalpha1,3(Fucalpha1,2)Galbeta1,4GlcNAc (MOAs preferred ligand) or without fucosylation in the xenotransplantation epitope. MOA has been co-crystallized with the linear blood group B trisaccharide Galalpha1,3Galbeta1,4GlcNAc using the hanging-drop vapour-diffusion technique at room temperature. MOA crystals were grown in the presence of ammonium formate and HEPES buffer. A 3.0 A data set has been collected. Preliminary analysis of the X-ray data is consistent with space group P3(1) or P3(2) and unit-cell parameters a = b = 105, c = 113 A, with two dimers per asymmetric unit.

Structure of the Fab fragment of the anti-murine EGFR antibody 7A7 and exploration of its receptor binding site

The EGF receptor is an important target of cancer immunotherapies. The 7A7 monoclonal antibody has been raised against the murine EGFR, but it cross-reacts with the human receptor. The results from experiments using immune-competent mice can therefore, in principle, be extrapolated to the corresponding scenario in humans. In this work we report the crystal structure of the 7A7 Fab at an effective resolution of 1.4Å. The antibody binding site comprises a deep pocket, located at the interface between the light and heavy chains, with major contributions from CDR loops H1, H2, H3 and L1. Binding experiments show that 7A7 recognizes a site on the EGFR extracellular domain that is not accessible in its most stable conformations, but that becomes exposed upon treatment with a tyrosine kinase inhibitor. This suggests a recognition mechanism similar to that proposed for mAb 806.

Crystal structure of the double azurin mutant Cys3Ser/Ser100Pro from Pseudomonas aeruginosa at 1.8 Å resolution: its folding–unfolding energy and unfolding kinetics

Azurin is a cupredoxin, which functions as an electron carrier. Its fold is dominated by a beta-sheet structure. In the present study, azurin serves as a model system to investigate the importance of a conserved disulphide bond for protein stability and folding/unfolding. For this purpose, we have examined two azurin mutants, the single mutant Cys3Ser, which disrupts azurins conserved disulphide bond, and the double mutant Cys3Ser/Ser100Pro, which contains an additional mutation at a site distant from the conserved disulphide. The crystal structure of the azurin double mutant has been determined to 1.8 A resolution(2), with a crystallographic R-factor of 17.5% (R(free)=20.8%). A comparison with the wild-type structure reveals that structural differences are limited to the sites of the mutations. Also, the rates of folding and unfolding as determined by CD and fluorescence spectroscopy are almost unchanged. The main difference to wild-type azurin is a destabilisation by approximately 20 kJ x mol(-1), constituting half the total folding energy of the wild-type protein. Thus, the disulphide bond constitutes a vital component in giving azurin its stable fold.

Crystallization and preliminary crystallographic analysis of the NAD(H)-binding domain of Escherichia coli transhydrogenase

Transhydrogenase is a proton-pumping membrane protein that is required for the cellular regeneration of NADPH. The NAD(H)-binding domain (domain I) of transhydrogenase from Escherichia coli was crystallized using the hanging-drop vapour-diffusion technique at room temperature. The crystals, which were grown from PEG 4000 and ammonium acetate in citrate buffer, belong to the triclinic space group P1, with unit-cell parameters a = 38.8, b = 66.8, c = 76.4 A, alpha = 67.5, beta = 80.8, gamma = 81.5 degrees. X-ray diffraction data were collected to 1.9 A resolution using synchrotron radiation. The crystals contain one dimer of transhydrogenase domain I per asymmetric unit.