Preliminary joint neutron time-of-flight and X-ray crystallographic study of human ABO(H) blood group A glycosyltransferase.

The biosyntheses of oligosaccharides and glycoconjugates are conducted by glycosyltransferases. These extraordinarily diverse and widespread enzymes catalyze the formation of glycosidic bonds through the transfer of a monosaccharide from a donor molecule to an acceptor molecule, with the stereochemistry about the anomeric carbon being either inverted or retained. Human ABO(H) blood group A α-1,3-N-acetylgalactosaminyltransferase (GTA) generates the corresponding antigen by the transfer of N-acetylgalactosamine from UDP-GalNAc to the blood group H antigen. To understand better how specific active-site-residue protons and hydrogen-bonding patterns affect substrate recognition and catalysis, neutron diffraction studies were initiated at the Protein Crystallography Station (PCS) at Los Alamos Neutron Science Center (LANSCE). A large single crystal was subjected to H/D exchange prior to data collection and time-of-flight neutron diffraction data were collected to 2.5 Šresolution at the PCS to ∼85% overall completeness, with complementary X-ray diffraction data collected from a crystal from the same drop and extending to 1.85 Šresolution. Here, the first successful neutron data collection from a glycosyltransferase is reported.

High-resolution study of the three-dimensional structure of horse heart metmyoglobin.

The three-dimensional structure of horse heart metmyoglobin has been refined to a final R-factor of 15.5% for all observed data in the 6.0 to 1.9 A resolution range. The final model consists of 1242 non-hydrogen protein atoms, 154 water molecules and one sulfate ion. This structure has nearly ideal bonding and bond angle geometry. A Luzzati plot of the variation in R-factor with resolution yields an estimated mean co-ordinate error of 0.18 A. An extensive analysis of the pattern of hydrogen bonds formed in horse heart metmyoglobin has been completed. Over 80% of the polypeptide chain is involved in eight helical segments, of which seven are composed mainly of alpha-helical (3.6(13))-type hydrogen bonds; the remaining helix is composed entirely of 3(10) hydrogen bonds. Altogether, of 102 hydrogen bonds between main-chain atoms only six are not involved in helical structures, and four of these six occur within beta-turns. The majority of water molecules in horse heart metmyoglobin are found in solvent networks that range in size from two to 35 members. The size of water molecule networks can be rationalized on the basis of three factors: the number of hydrogen bonds to the protein surface, the presence of charged side-chain atoms, and the ability to bridge to neighboring molecules in the crystal lattice. Bridging water networks form the dominant intermolecular interactions. The backbone conformation of horse heart metmyoglobin is very similar to sperm whale metmyoglobin, with significant differences in secondary structure occurring only near residues 119 and 120, where residues 120 to 123 in sperm whale form a distorted type I reverse turn and the horse heart protein has a type II turn at residues 119 to 122. Nearly all of the hydrogen bonds between main-chain atoms (occurring mainly in helical regions) are common to both proteins, and more than half of the hydrogen bonds involving side-chain atoms observed in horse heart are also found in sperm whale metmyoglobin. Unlike sperm whale metmyoglobin, the heme iron atom in horse heart metmyoglobin is not significantly displaced from the plane of the heme group.

Refinement of recombinant oncomodulin at 1.30 A resolution.

A refinement of the oncomodulin crystal structure at 1.30 A resolution has been carried out with X-ray data from the recombinant protein. The crystallographic R-factor values are 0.169 for 19,995 reflections in the range 6.0 to 1.30 A, which were used for the restrained least-squares refinement, and 0.176 for 20,186 observed reflections in the range 10.0 to 1.30 A. This high resolution refinement has enabled us to make more definitive statements about the molecular structure than was possible heretofore. The present model includes residues 1 to 108, the two Ca2+ of the CD and EF loops, two intermolecular Ca2+, and 103 water molecules per oncomodulin molecule. The electron density maps indicate disordered orientations for ten residues on the hydrophilic surface of the molecule. The pattern of molecular aggregation via intermolecular Ca2+, which occurs in the native rat oncomodulin structure, is also present in the recombinant oncomodulin structure. The Cys18 side-chain is not in a position that would be easily accessible for molecular dimerization via a disulphide bond. The substitution of Glu59, which is preserved in all the determined species of parvalbumin, by Asp59 in oncomodulin seems to break a stabilizing hydrogen bond in the CD loop and render the main-chain in positions 59 to 60 somewhat unstable. This instability in the CD loop, and the strong tendency of oncomodulin for molecular aggregation via intermolecular Ca2+, appear to be the two outstanding features that may account for oncomodulin's biological peculiarities.

Exploring the mimicry of polysaccharide antigens by anti-idiotypic antibodies. The crystallization, molecular replacement, and refinement to 2.8 A resolution of an idiotope-anti-idiotope Fab complex and of the unliganded anti-idiotope Fab.

The monoclonal antibody YsT9.1 is specific for the lipopolysaccharide A antigen of Brucella abortus. A complex formed between the Fab of YsT9.1 (Ab1) and the Fab of its antidiotopic monoclonal antibody T91AJ5 (Ab2) has been crystallized, and data have been collected to 2.8 A resolution. The space group is monoclinic P2(1), with one molecule per asymmetric unit. The structure was solved using a limited Patterson-correlation search over the asymmetric-unit of rotation space, and has been refined to an R-factor of 0.174. The complex is a head-to-head dimer, with the contact between the two Fabs almost completely restricted to their hypervariable loops. The interactions between the two Fabs in the complex are dominated by tyrosine residues, not only in the formation of hydrogen bonds, but in their participation in an aromatic ring network that spans the two Fv domains. The anti-idiotope was found to be unable to carry an internal image of the antigen and induce polysaccharide-specific "anti-anti-idiotopes" (Ab3) because the polysaccharide binding cleft on the Ab1 is too narrow and deep to allow comprehensive contact with the binding site of Ab2. The antibody T91AJ5 therefore is a class gamma anti-idiotope. The contact surfaces of the two Fabs are highly complementary; however, they are distinctly different in character. Each Fab has two separate binding surfaces of approximately equal size, but while the two binding surfaces of Ab1 are partitioned between the light and heavy chains, the two binding surfaces of Ab2 are shared between the chains, with the heavy chain responsible for about 60% of the total binding area. The structure of the unliganded Fab of Ab2 has also been solved by molecular replacement, and refined to an R-factor of 0.152 at 2.8 A resolution. This Fab crystallizes in the orthorhombic space group P2(1)2(1)2(1), with one molecule per asymmetric unit. The second hypervariable loop of the heavy chain of the Ab2 Fab is observed to undergo a significant and necessary conformational rearrangement in going from the unliganded to complexed form, and thus complex formation is an example of "induced fit" of antigen to antibody. The unliganded Ab1 Fab packs in the standard head-to-tail fashion observed for other Fabs. This mode of packing is precluded in the complex, by its head-to-head nature, and it is found to pack in tilted layers with most intermolecular contacts made between adjacent Ab2 Fab molecules.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation, crystallization and preliminary diffraction analyses of human pancreatic alpha-amylase.

Human pancreatic alpha-amylase has been isolated using a glycogen affinity precipitation procedure and crystallized in a form suitable for high resolution three-dimensional X-ray crystallographic analyses. Crystals are of the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 53.04 A, b = 74.80 A and c = 137.34 A, and contain only one protein molecule per asymmetric unit. Diffraction data have been collected and found to extend to 1.6 A resolution. These studies form the basis for elucidating the full atomic structure of human pancreatic alpha-amylase and thereby providing insight into the catalytic mechanism of this enzyme.

Antibody recognition of a conformational epitope in a peptide antigen: Fv-peptide complex of an antibody fragment specific for the mutant EGF receptor, EGFRvIII.

Epitope mapping studies and the determination of the structure to 1.8 A resolution have been carried out for the antigen-binding fragment MR1 in complex with peptide antigen. MR1 is specific for the novel fusion junction of the mutant epidermal growth factor receptor EGFRvIII and has been reported to have a high degree of specificity for the mutant EGFRvIII over the wild-type EGF receptor. The structure of the complex shows that the peptide antigen residue side-chains found by epitope mapping studies to be critical for recognition are accommodated in pockets on the surface of the Fv. However, the most distinctive portion of the peptide antigen, the novel fusion glycine residue, makes no contact to the Fv and does not contribute directly to the epitope. The specificity of MR1 lies in the ability of this glycine residue to assume the restricted conformation needed to form a type II' beta-hairpin turn more easily, and demonstrates that a peptide antigen can be used to generate a conformational epitope.

Effects of manno-1-deoxynojirimycin and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine on N-linked oligosaccharide processing in intestinal epithelial cells.

The effects of manno-1-deoxynojirimycin (ManDJN) and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP) were compared in IEC-6 intestinal epithelial cells in culture. ManDJN caused complete inhibition of N-linked complex oligosaccharide synthesis whereas a maximum of 80% inhibition was obtained with DMDP. HPLC showed similar endo H-sensitive oligosaccharides for control and treated cells. ManDJN caused a large increase in the levels of labeled Man7-9 GlcNAc and a decrease in Man5GlcNAc. DMDP produced similar changes except that the increase in Man7-9GlcNAc was less pronounced and some increase in glucosylated oligosaccharides was observed. Since the major oligosaccharides found in DMDP-treated cells were non-glucosylated, its primary effect on complex oligosaccharide synthesis is not due to inhibition of glucosidases, in contrast to what has been reported for influenza virus-infected MDCK cells [(1984) J. Biol. Chem. 259, 12409-12413].

Molluscicidal activity in the seeds of Millettia thonningii (Leguminosae: Papilionoideae).

Seeds of the leguminous plant Millettia thonningii were shown to possess promising molluscicidal activity against Bulinus trunctatus. The size of the snails was an important determinant of their susceptibility to the molluscicide; specimens with shell lengths of 2 to 3 mm being more susceptible than snails 5 to 6 mm long.

Seasonal variation in cytochrome P450 immunopositive protein levels, lipid peroxidation and genetic toxicity in digestive gland of the mussel Mytilus edulis.

The relationship between cytochrome P450 1A- and 2E-immunopositive proteins, lipid peroxidation and DNA strand breaks (SBs) was studied in Mytilus edulis digestive gland at different seasons and at different sites around the UK coast. Cytochrome P4501A (CYP1A)-immunopositive protein and DNA strand breaks were generally lowest in December but there was no correlation between PAH exposure (indicated by chemical measurement and CYP1A-immunopositive protein expression) and DNA strand breaks which was highest at the relatively non-polluted site (Port Quin). As with CYP1A, CYP2E1-immunopositive protein was maximal at most sites in May. Lipid peroxidation, in contrast, did not alter markedly throughout the year. In conclusion, DNA strand breakage was not correlated with any of the above parameters although it did correlate with "scope for growth" as did the inverse of PAH levels. The study highlights the need to establish the relative contribution of DNA damage and DNA repair processes to the production of DNA strand breaks and emphasises the need to consider seasonal variation in interpretation of biomarkers.

QSARs for the sublethal responses of marine mussels (Mytilus edulis).

The marine environment is contaminated with many organic compounds, some of which induce deleterious responses in biota. Biological impact can be assessed by measuring the physiological responses of mussels, though the task of establishing which of the bioaccumulated compounds cause the observed effects is complex. To facilitate this task, quantitative structure-activity relationships (QSARs) for the physiological responses are being established. In this paper, the responsiveness of ciliary feeding to alkanes and benzene-substituted alkanes is described and compared with a QSAR established previously for aromatic compounds. Most of the test compounds with aqueous solubilities greater than 70 micrograms dm-3 were toxic to feeding activity when bioaccumulated to similar concentrations, whereas compounds of lower solubility were less toxic. The only exceptions were the polyaromatic hydrocarbons pyrene and fluoranthene, which were less toxic than predicted from their solubility. These data are consistent with the hypothesis that the toxicity cut-off is due to solubility-related phenomena, the effect perhaps being enhanced for aromatic hydrocarbons dosed near to their solubility limits, by sequestration of crystals within the mussel tissues. These observations indicate that many organic contaminants detected by chemical analysis of mussels have no direct effect on filter feeding, whereas the less frequently determined volatile compounds are toxic.

SETOR: hardware-lighted three-dimensional solid model representations of macromolecules.

SETOR is designed to exploit the hardware lighting capabilities of the IRIS-4D series graphics workstations to render high-quality raster images of macromolecules that can undergo rotation and translation interactively. SETOR can render standard all-atom and backbone models of proteins or nucleic acids, but focuses on displaying protein molecules by highlighting elements of secondary structure. The program has a very friendly user interface that minimizes the number of input files by allowing the user to interactively edit parameters, such as colors, lighting coefficients, and descriptions of secondary structure via mouse activated dialogue boxes. The choice of polymer chain representation can be varied from standard vector models and van der Waal models, to a B-spline fit of polymer backbones that yields a smooth ribbon that approximates the polymer chain, to strict Cardinal splines that interpolate the smoothest curve possible that will precisely follow the polymer chain. The program provides a photograph mode, save/restore facilities, and efficient generation of symmetry-related molecules and packing diagrams. Additionally, SETOR is designed to accept commands and model coordinates from the standard input stream, and to control standard output. Ancillary programs provide a method to interactively edit hardcopy plots of all vector and many solid models generated by SETOR, and to produce standard HPGL or PostScript files. Examples of figures rendered by SETOR of a number of macromolecules of various classes are presented.

Characterization of protein-glycolipid recognition at the membrane bilayer.

A growing number of important molecular recognition events are being shown to involve the interactions between proteins and glycolipids. Glycolipids are molecules in which one or more monosaccharides are glycosidically linked to a lipid moiety. The lipid moiety is generally buried in the cell membrane or other bilayer, leaving the oligosaccharide moiety exposed but in close proximity to the bilayer surface. This presents a unique environment for protein-carbohydrate interactions, and studies to determine the influence of the bilayer on these phenomena are in their infancy. One important property of the bilayer is the ability to orient and cluster glycolipid species, as strong interactions in biological systems are often achieved through multivalency arising from the simultaneous association of two or more proteins and receptors. This is especially true of protein-carbohydrate binding because of the unusually low affinities that characterize the monovalent interactions. More recent studies have also shown that the composition of the lipid bilayer is a critical parameter in protein-glycolipid recognition. The fluidity of the bilayer allows for correct geometric positioning of the oligosaccharide head group relative to the binding sites on the protein. In addition, there are activity-based and structural data demonstrating the impact of the bilayer microenvironment on the modulation of oligosaccharide presentation. The use of model membranes in biosensor-based methods has supplied decisive evidence of the importance of the membrane in receptor presentation. These data can be correlated with three-dimensional structural information from X-ray crystallography, NMR, and molecular mechanics to provide insight into specific protein-carbohydrate inter--actions at the bilayer.

Horse heart metmyoglobin. A 2.8-A resolution three-dimensional structure determination.

The structure of horse heart metmyoglobin has been determined with a molecular replacement approach and subsequently refined using rigid body and restrained-parameter least squares methods to a conventional crystallographic R-factor of 0.16 for all observed reflections in the 6.0-2.8-A resolution range. The polypeptide chain of this protein is found to be organized into eight helical regions (labeled A-H) which collectively form a hydrophobic pocket in which the heme prosthetic group is bound. Our results show that the overall thermal motions of individual residues of horse heart metmyoglobin are correlated with their mean distances from the heme group. In comparisons with the structure of sperm whale metmyoglobin it has been found that horse heart metmyoglobin has unique polypeptide chain conformations in four regions. These include residues in the immediate vicinity of the amino and carboxyl termini, residues about Lys-16, and residues 117-124 which are in the interhelical region between helices G and H. Many of these conformational changes appear to occur as a consequence of a different pattern of salt-bridging interactions between charged residues on the surface of horse heart metmyoglobin. The overall average positional deviation observed between corresponding alpha-carbons in the polypeptide chains of horse heart and sperm whale metmyoglobin is 0.50 A. This value for atoms of the porphyrin core of the central heme group is 0.39 A. A total of 12 well defined water molecules and 1 sulfate ion are included in the current structural model of horse heart metmyoglobin. One of these water molecules is found to be coordinated to the heme iron atom and hydrogen bonded to the side chain of His-64. The sulfate ion is hydrogen bonded to amide groups at the amino-terminal end of the E-helix and, as well, forms similar interactions with the amino-terminal end of the D-helix of an adjacent protein molecule in the crystalline lattice.

Three-dimensional structure of cyanomet-sulfmyoglobin C.

The atomic structure of horse heart cyanomet-sulfmyoglobin C has been established by x-ray crystallographic techniques to a resolution of 2.0 A with an R value of 0.129. The protoheme IX prosthetic group of this thermodynamically stable sulfmyoglobin derivative has been converted to a chlorin in which the pyrrole ring bearing the 4-vinyl group is saturated and possesses an exocyclic thiolene ring. This study provides the three-dimensional structure of a protein with an iron-chlorin prosthetic group. The overall conformation of the surrounding polypeptide chain of the modified protein is very similar to that of the native protein. However, the addition of the sulfur atom has caused a distortion of the prosthetic group from that in the native protein to result in the repositioning of the side chains of some residues in the heme pocket.

1.70 A resolution structure of myoglobin from yellowfin tuna. An example of a myoglobin lacking the D helix.

The crystal structure of metmyoglobin from yellowfin tuna (Thunnus albacares) has been determined by molecular replacement methods and refined to a conventional R factor of 0.177 for all observed reflections in the range of 6.0-1.70 A resolution. Like other myoglobins for which a high-resolution structure is available, the polypeptide chain is organized into several helices that cooperate to form a hydrophobic pocket into which the heme prosthetic group is non-covalently bound; however, the D helix observed in other myoglobins is absent in myoglobin from yellowfin tuna and has been replaced with a random coil. As well, the A helix has a pronounced kink due to the presence of Pro16. The differences in structure between this and sperm whale myoglobin can be correlated with their reported dioxygen affinity and dissociation. The structure is in agreement with reported fluorescence data which show an increased Trp14.heme distance in yellowfin tuna compared to sperm whale myoglobin.

Experiments in microgravity: a comparison of crystals of a carbohydrate-binding fab grown on the ground, on space shuttle Discovery and on space station Mir.

The Fab fragment of the hybridoma antibody (YsT9.1) specific to Brucella abortus has been crystallized on earth using both Linbro plates and ground-based models of the flight hardware, as well as in microgravity on board the space shuttle Discovery and the space station Mir. Large-scale experiments using Linbro plates gave two different crystal morphologies, pyramidal and rhomboid, depending on conditions. The pyramidal crystals proved to scatter X-rays to higher resolution, and conditions within the ground-based flight hardware for both Discovery and Mir were adjusted to produce crystals with this morphology. The experiment on Discovery produced large crystals in each of ten chambers. The experiment on Mir produced crystals in only one of the five assigned chambers, despite the fact that the simultaneous ground-based experiment produced large crystals in every corresponding chamber. Data collection was attempted for crystals from both space and ground-based experiments. Higher resolution data was obtained from crystals grown on Discovery than from either Mir or ground-based crystals, even though the crystals obtained from Discovery were smaller and forced to grow over a much shorter period of time because of the shorter length of the shuttle mission.

The pyrrolidine alkaloid, 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, inhibits glycoprotein processing.

2,5-Dihydroxymethyl-3,4-dihydroxypyrrolidine (DMDP) is a pyrrolidine alkaloid that was isolated from the plant, Lonchocarpus sericeus. In the present study, DMDP was tested as an inhibitor of glycoprotein processing. MDCK cells were infected with influenza virus and the virus was raised in the presence of various amounts of DMDP. The glycoproteins were labeled by the addition of [2-3H]mannose or [1-3H]galactose to the medium. The virus was isolated by differential centrifugation and treated with Pronase to obtain glycopeptides. These glycopeptides were isolated by chromatography on Bio-Gel P-4, then digested with endoglucosaminidase H (Endo H) and rechromatographed on the Bio-Gel P-4 column. In the control virus, more than 70% of the glycopeptides were resistant to Endo H and were previously characterized as complex types of oligosaccharides. The remaining 20-25% are sensitive to Endo H and are of the high-mannose type. However, in the presence of DMDP (250 micrograms/ml), more than 80% of the glycopeptides are susceptible to digestion by Endo H. The oligosaccharide released by this treatment sized like a hexose11-12GlcNAc on a calibrated column of Bio-Gel P-4, and was only slightly susceptible to alpha-mannosidase treatment. This oligosaccharide was also labeled in the glucose moieties by growing the virus in [1-3H]galactose in the presence of DMDP. Following isolation, the oligosaccharide was subjected to complete methylation. Acid hydrolysis of the methylated oligosaccharide gave three methylated glucose derivatives, corresponding to 2,3,4,6-tetramethylglucose, 3,4,6-trimethylglucose, and 2,4,6-trimethylglucose in almost equal amounts. These data indicate that the oligosaccharide is a Glc3Man8-9-GlcNAc and that DMDP inhibits glucosidase I. Similar results were obtained with the cellular glycoproteins. DMDP did not inhibit the incorporation of [3H]leucine into protein in MDCK cells, nor did it inhibit virus production as measured by plaque counts or hemagglutination assays. DMDP did cause some inhibition of mannose incorporation into the lipid-linked monosaccharides, but incorporation into lipid-linked oligosaccharides was not greatly affected, and incorporation into protein was stimulated. These results suggest that the pyrrolidine alkaloids are a new class of processing inhibitors.

Donor substrate specificity of recombinant human blood group A, B and hybrid A/B glycosyltransferases expressed in Escherichia coli.

The human blood group A and B glycosyltransferases catalyze the transfer of GalNAc and Gal, to the (O)H-precursor structure Fuc alpha (1-2)Gal beta-OR to form the blood group A and B antigens, respectively. Changing four amino acids (176, 235, 266 and 268) alters the specificity from an A to a B glycosyltransferase. A series of hybrid blood group A/B glycosyltransferases were produced by interchanging these four amino acids in synthetic genes coding for soluble forms of the enzymes and expressed in Escherichia coli. The purified hybrid glycosyltransferases were characterized by two-substrate enzyme kinetic analysis using both UDP-GalNAc and UDP-Gal donor substrates. The A and B glycosyltransferases were screened with other donor substrates and found to also utilize the unnatural donors UDP-GlcNAc and UDP-Glc, respectively. The kinetic data demonstrate the importance of a single amino acid (266) in determining the A vs. B donor specificity.

Location of the active site of the bean alpha-amylase inhibitor and involvement of a Trp, Arg, Tyr triad.

Seeds of the common bean contain three homologous proteins: phytohaemagglutinin E, phytohaemagglutinin L and the lectin-like protein alpha-amylase inhibitor (alpha AI). Whereas the active site of lectins has been studied in great detail, there is no information on the active site of the related protein alpha AI, which exerts its biological activity by making a 1:1 complex with alpha-amylase. alpha-amylase inhibitor is synthesized as a 30 kDa precursor glycoprotein that needs to be processed at Asn77 to form an active molecule. Comparison of the amino acid sequence of the bean alpha AI with that of the bacterial amylase inhibitor, tendamistat, suggested that a region around Trp188 might be involved in the inhibitory site. When a three-dimensional model of the bean alpha AI was constructed based on its homology to the legume lectins, this Trp region was alongside Asn77. To test this site hypothesis, mutants of alpha AI were created by site-directed mutagenesis of the cDNA and expressed in transgenic tobacco. The mutant proteins R74N and WSY188-190GNV, as well as the double mutant, were inactive as inhibitors. These findings suggest that the active site of alpha AI consists of W188, R74 and Y190, in analogy to the Trp-Arg-Tyr motif of tendamistat, and that the processing of the polypeptide at Asn77 may be necessary to bring these residues in close proximity.

Crystallization and preliminary X-ray diffraction analysis of two homologous antigen-binding fragments in complex with different carbohydrate antigens.

The antigen-binding fragments (Fab) of two murine monoclonal antibodies (mAb) S25-2 and S45-18, specific for carbohydrate epitopes in the lipopolysacchaide of the bacterial family Chlamydiaceae, have been crystallized in the presence and absence of synthetic oligosaccharides corresponding to their respective haptens. Crystals of both Fabs show different morphology depending on the presence of antigens. The sequence of mAb S45-18 was determined and shows a remarkable homology to that reported for mAb S25-2. These crystals offer an unparalleled opportunity to compare the structure and modes of binding of two homologous antibodies to similar but distinct carbohydrate epitopes.