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R C Garratt

Publications and source records attributed to R C Garratt.

At least 37 records · Page 2Linked to original sources
PubMed1997

Characterization of a Schistosoma mansoni gene encoding a homologue of the Y-box binding protein.

We have cloned and characterized a Schistosoma mansoni cDNA encoding a basic protein homologous to the human Y-box binding protein 1 (YB-1). The 1.3-kb S. mansoni YB-1 transcript, which was shown to be expressed in various stages of the parasite life cycle, codes for a protein of 217 amino acids containing, towards its N-terminus, a nucleic acid binding motif, known as the cold-shock domain (CSD). This domain is 64% identical to the cold-shock domain of other members of the Y-box binding protein family and 43% identical to the cold-shock protein CspA of Escherichia coli. In S. mansoni YB-1, the cold-shock domain possess some structural characteristics that permit dimer formation as occurs in the Bacillus subtilis cold-shock protein CspB. The C-terminal region of S. mansoni YB-1 differs from the other Y-box binding proteins because of the presence of tandem repeats of Arg and Gly, suggesting the formation of a fibroin-like beta-sandwich structure. This novel folding pattern for the C-terminus of S. mansoni YB-1 might suggest a distinct specific function for this protein in the parasite.

Amino Acid Sequence↗
PubMed1997

Crystallization and preliminary crystallographic studies of calgranulin C, an S100-like calcium-binding protein from pig granulocytes.

Calgranulin C (CAGC) from pig granulocytes has been crystallized and X-ray diffraction data have been collected to 2.6 A resolution. The crystals belong to the trigonal system, space group P3(1)21 or P3(2)21, cell parameters a = b = 54.35 (2), c = 141.32 (5) A and probably contain two molecules in the asymmetric unit. CAGC is amongst the first reported typical S100-1ike calcium-binding protein to be crystallized and studied by X-ray crystallography.

Journal Article↗
PubMed1997

Crystallization and preliminary crystallographic data of a neutrophil migration-inducing lectin (KM+) extracted from the seed of Artocarpus integrifolia.

The tetrameric KM+ lectin from the seeds of Artocarpus integrifolia has, when compared to other plant lectins, the singular property of directly inducing neutrophil migration into the peritoneal cavity or into the air pouch of rats. This protein crystals have been grown and they belong to the orthorhombic system with space group C222(1). The unit cell parameters are a = 54.4 A, b = 127.9 A and c = 99.8 A. A native diffraction dataset to 2.8 A was collected and an analysis of the self-rotation function has shown the presence of only one independent non-crystallographic 2-fold axis orthogonal to the crystal b-axis, compatible with a dimer in the asymmetric unit.

Animals↗
PubMed1996

A Schistosoma mansoni fatty acid-binding protein, Sm14, is the potential basis of a dual-purpose anti-helminth vaccine.

Molecular cloning of components of protective antigenic preparations has suggested that related parasite fatty acid-binding proteins could form the basis of the protective immune crossreactivity between the parasitic trematode worms Fasciola hepatica and Schistosoma mansoni. Molecular models of the two parasite proteins showed that both molecules adopt the same basic three-dimensional structure, consisting of a barrel-shaped molecule formed by 10 antiparallel beta-pleated strands joined by short loops, and revealed the likely presence of crossreactive, discontinuous epitopes principally derived from amino acids in the C-terminal portions of the molecules. A recombinant form of the S. mansoni antigen, rSm14, protected outbred Swiss mice by up to 67% against challenge with S. mansoni cercariae in the absence of adjuvant and without provoking any observable autoimmune response. The same antigen also provided complete protection against challenge with F. hepatica metacercariae in the same animal model. The results suggest that it may be possible to produce a single vaccine that would be effective against at least two parasites, F. hepatica and S. mansoni, of veterinary and human importance, respectively.

Amino Acid Sequence↗
PubMed1996

Novel features of serine protease active sites and specificity pockets: sequence analysis and modelling studies of glutamate-specific endopeptidases and epidermolytic toxins.

With a view to obtaining a better understanding of the structural determinants of P1 glutamate specificity in glutamate-specific endopeptidases (GSEs), the active sites and specificity pockets of such enzymes from Bacillus licheniformis (gse-bl), Bacillus subtilis (mpr) and Staphylococcus aureus (v8 protease) were modelled. This approach was extended to the epidermolytic toxins (ETs), responsible for the staphylococcal scalded skin syndrome. We identify a canonical structure for the S1 subsite, composed of H213 and T190, both of which we predict to interact directly with the P1 glutamate. The possible importance of R30 (for gse-bl and mpr) and of the N-terminus (for gse-bl, mpr and v8 protease) was also examined. In the case of mpr, a G193C substitution is predicted to participate in a novel disulphide bridge which stabilizes C193 in such a way as to maintain the oxyanion hole. In v8, the loss or substitution of several important structural components around D102 of the catalytic triad probably explains its reduced catalytic efficiency in comparison with other GSEs. In the case of the epidermolytic toxins K216 may be important for the previously reported phospholipase C-like activity, since the model predicts that it may stabilize the negative charge on the phosphonyl group.

Algorithms↗
PubMed1995

Structure and catalytic mechanism of glucosamine 6-phosphate deaminase from Escherichia coli at 2.1 A resolution.

BACKGROUND: Glucosamine 6-phosphate deaminase from Escherichia coli is an allosteric hexameric enzyme which catalyzes the reversible conversion of D-glucosamine 6-phosphate into D-fructose 6-phosphate and ammonium ion and is activated by N-acetyl-D-glucosamine 6-phosphate. Mechanistically, it belongs to the group of aldoseketose isomerases, but its reaction also accomplishes a simultaneous amination/deamination. The determination of the structure of this protein provides fundamental knowledge for understanding its mode of action and the nature of allosteric conformational changes that regulate its function. RESULTS: The crystal structure of glucosamine 6-phosphate deaminase with bound phosphate ions is presented at 2.1 A resolution together with the refined structures of the enzyme in complexes with its allosteric activator and with a competitive inhibitor. The protein fold can be described as a modified NAD-binding domain. CONCLUSIONS: From the similarities between the three presented structures, it is concluded that these represent the enzymatically active R state conformer. A mechanism for the deaminase reaction is proposed. It comprises steps to open the pyranose ring of the substrate and a sequence of general base-catalyzed reactions to bring about isomerization and deamination, with Asp72 playing a key role as a proton exchanger.

Aldose-Ketose Isomerases↗
PubMed1994

Characterization and structural analysis of a functional human serum transferrin variant and implications for receptor recognition.

The nucleotide and amino acid substitutions leading to the only known functional variant of human serum transferrin have been characterized by sequencing of a peptide produced by cyanogen bromide digestion and genomic PCR coupled with cycle sequencing, respectively. There is an amino acid substitution at position 394 (Gly-->Arg) resulting from a mutational transition, G-->A, in the first nucleotide of the codon GGG. The Zn(2+)-, Al(3+)-, and Cu(2+)-binding properties of the variant, ascertained by UV difference spectra and, in the case of copper, protein fluorescence quenching, confirm that these metals binds to only one of the two sites. Solution X-ray scattering measurements indicate that the lobe (the C-lobe) containing the mutation remains "open" in the iron-bound state, and modeling studies suggest that this is a consequence of the formation of a salt bridge between Arg394 in the variant protein and Asp392, one of the iron-binding ligands in the C-lobe. This rationalizes for the first time the observed reduction in receptor affinity of the diferric variant protein for PHA-stimulated lymphocytes [Young, S.P., et al. (1984) Br. J. Haematol. 56, 581-587] and here repeated with K562 cells. These data lend support to the hypothesis that the closed conformation for both lobes contributes to receptor recognition.

Aluminum↗
PubMed1993

A structural model for the glutamate-specific endopeptidase from Streptomyces griseus that explains substrate specificity.

We present a model for the three-dimensional structure of the glutamate-specific endopeptidase from Streptomyces griseus based on the crystal structures of other bacterial proteases of the trypsin family. For the first time a structural model is described which attempts to explain the basis of P1 glutamate specificity in serine proteases. Several important changes to the S1 pocket with respect to other members of the family of different specificity are described. Of particular interest is the presence of a histidine at position 213 and the substitution of Arg-138 by lysine. Other biochemical evidence concerning substrate preferences can be rationalized on the basis of the model.

Amino Acid Sequence↗
PubMed1993

The mechanism of iron uptake by transferrins: the structure of an 18 kDa NII-domain fragment from duck ovotransferrin at 2.3 A resolution.

The molecular structure of an iron-containing 18 kDa fragment of duck ovotransferrin, obtained by proteolysis of the intact protein, has been elucidated by protein crystallographic techniques at 2.3 A resolution. This structure supports a mechanism of iron uptake in the intact protein whereby the binding of the synergistic (bi)carbonate anion is followed by binding of the metal with the lobe in the open configuration. These stages are then followed by domain closure in which the aspartic acid residue plays a further key role, by forming an interdomain hydrogen-bond interaction in addition to serving as a ligand to the iron. This essential dual role is highlighted by model building studies on the C-terminal lobe of a known human variant. In this variant a mutation of a glycine by an arginine residue enables the aspartic acid to form an ion pair and reduce its effectiveness for both metal binding and domain closure. The X-ray structure of the 18 kDa fragment strongly suggests that the histidine residue present at the iron binding site of the intact protein and arising from the second interdomain connecting strand has been removed during the preparative proteolysis.

Journal Article↗
PubMed1992

Crystallization and preliminary crystallographic studies of glucosamine-6-phosphate deaminase from Escherichia coli K12.

Hexameric glucosamine-6-phosphate deaminase from Escherichia coli has been crystallized isomorphously with both phosphate and ammonium sulphate as precipitants, over a wide pH range (6.0 to 9.0). The crystals belong to space group R32 and the cell parameters in the hexagonal setting are a = b = 125.9 A and c = 223.2 A. A complete native data set was collected to 2.1 A resolution. Self-rotation function studies suggest that the hexamers sit on the 3-fold axis and have point group symmetry 32, with a non-crystallographic dyad relating two monomers linked by an interchain disulfide bridge. A possible packing for the unit cell is proposed.

Aldose-Ketose Isomerases↗
PubMed1992

A molecular model for the tumour-associated antigen, p97, suggests a Zn-binding function.

The primary structure of p97 (melanotransferrin) has been compared with other members of the transferrin superfamily. A molecular structure of p97 has been modelled based on the crystal structure of diferric rabbit serum transferrin. The most significant amino acid substitutions in p97 are almost exclusively limited to only two regions; the C-lobe iron-binding cleft and the interlobe contact region. The latter includes within the N-terminal lobe a Zn-binding consensus sequence found in metallopeptidases, and in the C-terminal lobe a glutamic acid residue (Glu-394) capable of completing a potential thermolysin-like Zn-binding site. Thus, p97 may have a Zn-binding potential, unique amongst the transferrin superfamily.

Amino Acid Sequence↗
PubMed1991

X.a.f.s. studies of chicken dicupric ovotransferrin.

A comparison of Cu K-edge x.a.f.s. spectra with that of the equivalent Fe K-edge for chicken ovotransferrin (COT) indicates that the metal ions occupy essentially the same binding sites in the protein. However, in the case of the Cu2+ complex the metal appears to have reduced co-ordination. Changes are observed in the x.a.f.s. of 90%-saturated COT (Cu1.8COT) on freeze-drying. The three-dimensional X-ray structures of rabbit serum transferrin and human lactoferrin have shown that the ferric cations are co-ordinated by four protein ligands and a bidentate carbonate anion in a distorted octahedral arrangement [Anderson, Baker, Dodson, Norris, Rumball, Waters & Baker (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1768-1774; Anderson, Baker, Norris, Rice and Baker (1989) J. Mol. Biol. 209, 711-734; Bailey, Evans, Garratt, Gorinsky, Hasnain, Horsburgh, Jhoti, Lindley, Mydin, Sarra & Watson (1988) Biochemistry 27, 5804-5812]. This structural information, together with the differences in e.x.a.f.s. spectra for solution and freeze-dried samples of diferric COT [Hasnain, Evans, Garratt & Lindley (1987) Biochem. J. 247, 369-375] suggests that the synergistic carbonate anion may be capable of behaving as a unidentate linkage to the Cu2+ in the dicupric complex. Data for Cu1.8COT are consistent with only three protein ligands bound to Cu2+, monodentate binding of the synergistic anion in one lobe and its bidentate binding in the other lobe. Such flexibility in the anion co-ordination may be a requirement for the uptake and release of metals by the transferrins.

Amino Acid Sequence↗
PubMed1991

An extension of secondary structure prediction towards the prediction of tertiary structure.

Secondary structure prediction parameters and optimised decision constants for use with the method of Garnier et al. [(1978) J. Mol. Biol. 120, 97-120] have been derived for two new and distinct substates of beta-structure. These we term internal and external on the basis of their hydrogen bonding patterns. The profiles of the amino acids for several of the parameters are considerably different in the two substates. Predictions using the new parameters attempt to distinguish the strands at the core of the beta-sheet from those at its edges and so restrict the possible topologies in tertiary structure prediction. The potential application of these parameters is illustrated for the class of beta/alpha proteins.

Adenylate Kinase↗
PubMed1988

Molecular structure of serum transferrin at 3.3-A resolution.

Serum transferrin is a metal-binding glycoprotein, molecular weight ca. 80,000, whose primary function is the transport of iron in the plasma of vertebrates. The X-ray crystallographic structure of diferric rabbit serum transferrin has been determined to a resolution of 3.3 A. The molecule has a beta alpha structure of similar topology to human lactoferrin and is composed of two homologous lobes that each bind a single ferric ion. Each lobe is further divided into two dissimilar domains, and the iron-binding site is located within the interdomain cleft. The iron is bound by two tyrosines, a histidine, and an aspartic acid residue. The location of the 19 disulfide bridges is described, and their possible structural roles are discussed in relation to the transferrin family of proteins. Mapping of the intron/exon splice junctions onto the molecule provides some topological evidence in support of the putative secondary role for transferrin in stimulating cell proliferation.

Animals↗
PubMed1988

Crystallization and preliminary analysis of an 18,000 Mr fragment of duck ovotransferrin.

Crystals of an 18,000 Mr iron-binding fragment of duck ovotransferrin, corresponding to domain II of the N-terminal lobe, have been obtained. The crystals belong to the trigonal system, P31 (or enantiomer) with a = b = 41.3(1) A, c = 81.2(2) A (1 A = 0.1 nm) and one molecule per asymmetric unit assuming a solvent content of 40% by volume. The crystals are stable at +4 degrees C and diffract to at least 2.3 A resolution.

Animals↗
PubMed1987

An extended-X-ray-absorption-fine-structure study of freeze-dried and solution ovotransferrin. Evidence for water co-ordination at the metal-binding sites.

Our previous extended-X-ray-absorption-fine-structure (e.x.a.f.s.) study has shown that the probable iron environment in chicken ovotransferrin involves two low-Z ligands (consistent with phenolate linkages) at 0.185(1) nm and four low-Z ligands at 0.204(1) nm [Garratt, Evans, Hasnain & Lindley (1986) Biochem. J. 233, 479-484]. Herein we provide additional information from the e.x.a.f.s. and near-edge structure suggestive of a decrease in the co-ordination number of ovotransferrin-bound iron upon freeze-drying. These effects are reversible, and exposure of the freeze-dried material to a humid atmosphere results in reversion to the solution spectra. Progressive rehydration was monitored by using e.p.r. spectroscopy and was confirmed by recording the high-resolution X-ray-absorption near-edge structure (x.a.n.e.s.). The results suggest the presence of a labile water molecule at the iron-binding sites of ovotransferrin in solution.

Animals↗
PubMed1986

An extended-X-ray-absorption-fine-structure investigation of diferric transferrins and their iron-binding fragments.

Iron K-edge extended-X-ray-absorption-fine-structure (e.x.a.f.s.) spectra were recorded for diferric human and rabbit serum transferrins and for diferric chicken ovotransferrin in aqueous solution; for ovotransferrin e.x.a.f.s. spectra from the N-terminal and C-terminal domain fragments were also measured. The overall spectral profiles closely resemble one another, indicating similar iron-binding sites. The simulation of the diferric ovotransferrin spectrum suggests a first co-ordination shell consisting of six low-Z ligands (nitrogen/oxygen), two ligands at a distance of approx. 0.185 nm (1.85 A) and four ligands at approx. 0.204 nm (2.04 A). The two shorter distances may correspond to Fe-O (tyrosine), whereas the longer distance is consistent with Fe-N (histidine) and Fe-O (water). Detailed analysis of the spectra of the N-terminal and C-terminal fragments indicates a difference in the short ligand distance.

Binding Sites↗