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J H Naismith

Publications and source records attributed to J H Naismith.

At least 37 records · Page 2Linked to original sources
PubMed2000

RmlC, the third enzyme of dTDP-L-rhamnose pathway, is a new class of epimerase.

Deoxythymidine diphosphate (dTDP)-L-rhamnose is the precursor of L-rhamnose, a saccharide required for the virulence of some pathogenic bacteria. dTDP-L-rhamnose is synthesized from glucose-1-phosphate and deoxythymidine triphosphate (dTTP) via a pathway involving four distinct enzymes. This pathway does not exist in humans and the enzymes involved in dTDP-L-rhamnose synthesis are potential targets for the design of new therapeutic agents. Here, the crystal structure of dTDP-6-deoxy-D-xylo-4-hexulose 3,5 epimerase (RmlC, EC5.1.3.13) from Salmonella enterica serovar Typhimurium was determined. The third enzyme of the rhamnose biosynthetic pathway, RmlC epimerizes at two carbon centers, the 3 and 5 positions of the sugar ring. The structure was determined by multiwavelength anomalous diffraction to a resolution of 2.17 A. RmlC is a dimer and each monomer is formed mainly from two beta-sheets arranged in a beta-sandwich. The structure of a dTDP-phenol-RmlC complex shows the substrate-binding site to be located between the two beta-sheets; this site is formed from residues of both monomers. Sequence alignments of other RmlC enzymes confirm that this region is very highly conserved. The enzyme is distinct structurally from other epimerases known and thus, is the first example of a new class of carbohydrate epimerase.

Amino Acid Sequence↗
PubMed2000

The purification, crystallization and preliminary structural characterization of glucose-1-phosphate thymidylyltransferase (RmlA), the first enzyme of the dTDP-L-rhamnose synthesis pathway from Pseudomonas aeruginosa.

Glucose-1-phosphate thymidylyltransferase (RmlA; E.C. 2.7.7.24) is the first of four enzymes involved in the biosynthesis of dTDP-L-rhamnose, the precursor of L-rhamnose, a key component of the cell wall of many pathogenic bacteria. RmlA catalyses the condensation of thymidine triphosphate (dTTP) and alpha-D-glucose-1-phosphate (G1P), yielding dTDP-D-glucose. RmlA from Pseudomonas aeruginosa has been overexpressed and purified. Crystals of the enzyme have been grown using the sitting-drop vapour-diffusion technique with PEG 6000 and lithium sulfate as precipitant. Several diffraction data sets of single frozen crystals were collected to a resolution of 1.66 A. Crystals belonged to space group P1, with unit-cell parameters a = 71.5, b = 73.1, c = 134.7 A, alpha = 89.9, beta = 80.9, gamma = 81.1 degrees. The asymmetric unit contains eight monomers in the form of two RmlA tetramers with a solvent content of 51%. Selenomethionine-labelled protein has been obtained and crystallized.

Chromatography, High Pressure Liquid↗
PubMed2000

The purification, crystallization and structural elucidation of dTDP-D-glucose 4,6-dehydratase (RmlB), the second enzyme of the dTDP-L-rhamnose synthesis pathway from Salmonella enterica serovar typhimurium.

dTDP-D-glucose 4,6-dehydratase (RmlB) is the second of four enzymes involved in the dTDP-L-rhamnose pathway and catalyzes the dehydration of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose. The ultimate product of the pathway, dTDP-L-rhamnose, is the precursor of L-rhamnose, which is a key component of the cell wall of many pathogenic bacteria. RmlB from Salmonella enterica serovar Typhimurium has been overexpressed and purified, and crystals of the enzyme have been grown using the sitting-drop vapour-diffusion technique with lithium sulfate as precipitant. Diffraction data have been obtained to a resolution of 2.8 A on a single frozen RmlB crystal which belongs to space group P2(1), with unit-cell parameters a = 111.85, b = 87.77, c = 145.66 A, beta = 131.53 degrees. The asymmetric unit contains four monomers in the form of two RmlB dimers with a solvent content of 62%. A molecular-replacement solution has been obtained and the model is currently being refined.

Bacterial Proteins↗
PubMed2000

Identification of conserved residues contributing to the activities of adenovirus DNA polymerase.

Adenovirus codes for a DNA polymerase that is a member of the DNA polymerase alpha family and uses a protein primer for initiation of DNA synthesis. It contains motifs characteristic of a proofreading 3'-5'-exonuclease domain located in the N-terminal region and several polymerase motifs located in the C-terminal region. To determine the role of adenovirus DNA polymerase in DNA replication, 22 site-directed mutations were introduced into the conserved DNA polymerase motifs in the C-terminal region of adenovirus DNA polymerase and the mutant forms were expressed in insect cells using a baculovirus expression system. Each mutant enzyme was tested for DNA binding activity, the ability to interact with pTP, DNA polymerase catalytic activity, and the ability to participate in the initiation of adenovirus DNA replication. The mutant phenotypes identify functional domains within the adenovirus DNA polymerase and allow discrimination between the roles of conserved residues in the various activities carried out by the protein. Using the functional data in this study and the previously published structure of the bacteriophage RB69 DNA polymerase (J. Wang et al., Cell 89:1087-1099, 1997), it is possible to envisage how the conserved domains in the adenovirus DNA polymerase function.

Adenoviridae↗
PubMed1999

A general method for co-crystallization of concanavalin A with carbohydrates.

A small grid of conditions has been developed for co-crystallization of the plant lectin concanavalin A (conA) and polysaccharides. Crystals have been obtained of complexes of conA with alpha1-2 mannobiose, 1-methyl alpha1-2 mannobiose, fructose, a trisaccharide and a pentasaccharide. The crystals diffract to resolutions of 1. 75-2.7 A using a copper rotating-anode source. The crystals are grown in the presence of polyethylene glycol 6K [10-20%(w/v)] at around pH 6.0. Optimization for each particular carbohydrate requires small adjustments in the conditions; however, all complexes give some crystalline precipitate in this limited grid. The alpha1-2 mannobiose complex crystals diffract to 1.75 A with space group I222 and cell dimensions a = 91.7, b = 86.8, c = 66.6 A. One monomer is present in the asymmetric unit. The 1-methyl alpha1-2 mannobioside complex crystallizes in space group P212121, cell dimensions a = 119. 7, b = 119.7, c = 68.9 A and diffract to 2.75 A. One tetramer is present in the asymmetric unit. Two crystal forms of the conA-fructose complex have been obtained. The first has space group P212121, cell dimensions a = 121.7, b = 119.9, c = 67.3 A with a tetramer in the asymmetric unit and diffracts to 2.6 A. The second crystallizes in space group C2221, cell dimensions a = 103.3, b = 117.9, c = 254.3 A with two dimers in the asymmetric unit and diffracts to 2.42 A. Structures and crystallization of the trisaccharide-conA and pentasaccharide-conA complexes have already been reported. In all complexes, the protein is found as a tetramer, although varying combinations of non-crystallographic and crystallographic symmetry are involved in generating the tetramer. The precise packing of the tetramer varies from crystal to crystal and it is likely that this variability facilitates crystallization.

Carbohydrate Sequence↗
PubMed1999

Man alpha1-2 Man alpha-OMe-concanavalin A complex reveals a balance of forces involved in carbohydrate recognition.

We have determined the crystal structure of the methyl glycoside of Man alpha1-2 Man in complex with the carbohydrate binding legume lectin concanavalin A (Con A). Man alpha1-2 Man alpha-OMe binds more tightly to concanavalin A than do its alpha1-3 and alpha1-6 linked counterparts. There has been much speculation as to why this is so, including a suggestion of the presence of multiple binding sites for the alpha1-2 linked disaccharide. Crystals of the Man alpha1-2 Man alpha-OMe-Con A complex form in the space group P2(1)2(1)2(1) with cell dimensions a = 119.7 A, b = 119.7 A, c = 68.9 A and diffract to 2. 75A. The final model has good geometry and an R factor of 19.6% (Rfree= 22.8%). One tetramer is present in the asymmetric unit. In three of the four subunits, electron density for the disaccharide is visible. In the fourth only a monosaccharide is seen. In one subunit the reducing terminal sugar is recognized by the monosaccharide site; the nonreducing terminal sugar occupies a new site and the major solution conformation of the inter-sugar glycosidic linkage conformation is adopted. In contrast, in another subunit the non reducing terminal sugar sits in the so called monosaccharide binding site; the reducing terminal sugar adopts a different conformation about its inter-sugar glycosidic linkage in order for the methyl group to access a hydrophobic pocket. In the third subunit, electron density for both binding modes is observed. We demonstrate that an extended carbohydrate binding site is capable of binding the disaccharide in two distinct ways. These results provide an insight in to the balance of forces controlling protein carbohydrate interactions.

Carbohydrate Conformation↗
PubMed1999

Initiating a crystallographic study of UDP-galactopyranose mutase from Escherichia coli.

UDP-galactopyranose mutase, the enzyme responsible for the conversion of UDP-galactopyranose to UDP-galactofuranose, has been crystallized in a form suitable for X-ray diffraction studies. UDP-galactofuranose is a key component of mycobacterial cell walls. Crystals of both the native protein and a selenomethionine variant have been grown by the vapour-diffusion method in hanging drops, and diffract to beyond 3.0 A using synchrotron radiation. Equilibration was against a solution of 20%(w/v) polyethylene glycol (4K), 12%(v/v) 2--propanol, 0.1 M HEPES pH 7.6 at 293.5 K. Crystals grow as thin plates of dimensions 0.4 x 0.2 x approximately 0.02 mm. They are monoclinic [corrected], space group P21, with unit-cell dimensions a = 71. 12, b = 58.42, c = 96.38 A, beta = 96.38 degrees. 92% (native) and 94% (selenomethionine) complete data sets have been recorded to 2.9 A (Rmerge = 5.0%) and 3.0 A (Rmerge = 6.9%), respectively. The Matthews coefficient is 2.35 A3 Da-1 for a dimer in the asymmetric unit, the solvent content being 47%. Diffraction data have also been recorded on a putative platinum derivative to 3.5 A.

Amino Acid Sequence↗
PubMed1999

Purification, crystallization and preliminary structural studies of dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase (RmlC), the third enzyme of the dTDP-L-rhamnose synthesis pathway, from Salmonella enterica serovar typhimurium.

L-Rhamnose is an essential component of the cell wall of many pathogenic bacteria. Its precusor, dTDP-L-rhamnose, is synthesized from alpha-D-glucose-1-phosphate and dTTP via a pathway requiring four distinct enzymes: RmlA, RmlB, RmlC and RmlD. RmlC was overexpressed in Escherichia coli. The recombinant protein was purified by a two-step protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated that the recombinant protein is monodisperse. Crystals were obtained using the sitting-drop vapour-diffusion method with ammonium sulfate as precipitant. Diffraction data were collected on a frozen crystal to a resolution of 2.17 A. The crystal belongs to either space group P3121 or P3221, with unit-cell parameters a = b = 71.56, c = 183.53 A and alpha = beta = 90, gamma = 120 degrees.

Carbohydrate Epimerases↗
PubMed1999

Initiating a structural study of 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli.

2-Keto-3-deoxy-6-phosphogluconate aldolase (KDPG aldolase, E.C. 4.1. 2.14) is a member of the pyruvate/phosphoenolpyruvate aldolase family. It is also a synthetically useful enzyme, capable of catalyzing the stereoselective aldol addition of pyruvate to a range of unnatural electrophilic substrates. The recombinant protein was purified by a two-step HPLC protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated the protein to be monodisperse. Crystals were obtained using the sitting-drop vapour-diffusion method, with PEG 6K as precipitant. Diffraction data were collected on a frozen crystal to a resolution of 2.26 A on station PX9.6 at the Daresbury synchrotron. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.2, b = 77.9, c = 146.8 A.

Aldehyde-Lyases↗
PubMed1999

Overexpression, purification, crystallization and preliminary structural study of dTDP-6-deoxy-L-lyxo-4-hexulose reductase (RmlD), the fourth enzyme of the dTDP-L-rhamnose synthesis pathway, from Salmonella enterica serovar Typhimurium.

L-Rhamnose is an essential component of the cell wall of many pathogenic bacteria. Its precursor, dTDP-L-rhamnose, is synthesized from alpha-D-glucose-1-phosphate and dTTP via a pathway requiring four distinct enzymes: RmlA, RmlB, RmlC and RmlD. RmlD catalyses the terminal step of this pathway by converting dTDP-6-deoxy-L-lyxo-4-hexulose to dTDP-L-rhamnose. RmlD from -Salmonella enterica serovar Typhimurium has been overexpressed in Escherichia coli. The recombinant protein was purified by a two--step protocol involving anion-exchange and hydrophobic chromatography. Dynamic light-scattering experiments indicated that the recombinant protein is monodisperse. Crystals of native and selenomethionine-enriched RmlD have been obtained using the sitting-drop vapour-diffusion method with polyethylene glycol as precipitant. Diffraction data have been collected from orthorhombic crystals of both native and selenomethionyl-derivatized protein, allowing tracing of the protein structure.

Carbohydrate Dehydrogenases↗
PubMed1998

Structure of ubiquitin-conjugating enzyme 9 displays significant differences with other ubiquitin-conjugating enzymes which may reflect its specificity for sumo rather than ubiquitin.

The three-dimensional structure of ubiquitin-conjugating enzyme 9 (Ubc9) has been obtained to a resolution of 2.8 A by molecular replacement followed by a combination of automated refinement and graphical intervention. Diffraction data were recorded on a single crystal in space group P43 with cell dimensions a = b = 73.9, c = 42. 9 A. The final model has an R factor of 21.3% for all data to 2.8 A. Only the N-terminal methionine, a two-residue N-terminal extension and a four-residue loop are not located by the final electron-density map. Ubc9 is now known to be the first sumo, a new ubiquitin-like protein, conjugating enzyme and does not conjugate ubiquitin. The structure of Ubc9 shows important differences compared with the structures of known ubiquitin-conjugating enzymes. At the N-terminal helix, the structural and sequence alignments are out of register by one amino acid giving Ubc9 a different recognition surface compared to ubiquitin-conjugating enzymes. This is coupled to a profound change in the electrostatic surface of the molecular face remote from the catalytic site. These differences may be important in recognition of other proteins in the Sumo conjugation pathway. The catalytic cysteine in Ubc9 has a positively charged lip and a negatively charged ridge nearby. Both these features seem confined to sumo-conjugating enzymes, and a sequence alignment of sumo and ubiquitin suggests how these might play a role in sumo/ubiquitin discrimination.

Amino Acid Sequence↗
PubMed1998

Crystallization of succinylated concanavalin A bound to a synthetic bivalent ligand and preliminary structural analysis.

Crystals have been obtained of succinylated concanavalin A complexed to a novel bidentate synthetic ligand. The crystals are the first example of a lectin with a synthetic multivalent ligand and the first report of crystallization of succinylated concanavalin A. The crystals were obtained by sitting-drop vapour diffusion equilibrating with a solution of 20% polyethylene glycol, pH 5, 293. 5 K. Crystals are orthorhombic, belonging to space group C2221 with unit-cell dimensions of a = 99.1, b = 127.4, c = 118.9 A. The asymmetric unit contains a dimer, with over 65% of the volume occupied by water. The ligand cross links concanavalin A monomers. Succinylated concanavalin A is known to be a dimer in solution, yet it is found as the typical concanavalin A tetramer in the crystal. The contacts holding together the tetramer appear extensive and suggest that a fine balance between dimer and tetramers exists. Data to 2.65 A have been collected and the structure determined by the molecular replacement method.

Azides↗
PubMed1998

The role of polar interactions in the molecular recognition of CD40L with its receptor CD40.

CD40 Ligand (CD40L) is transiently expressed on the surface of T-cells and binds to CD40, which is expressed on the surface of B-cells. This binding event leads to the differentiation, proliferation, and isotype switching of the B-cells. The physiological importance of CD40L has been demonstrated by the fact that expression of defective CD40L protein causes an immunodeficiency state characterized by high IgM and low IgG serum levels, indicating faulty T-cell dependent B-cell activation. To understand the structural basis for CD40L/CD40 association, we have used a combination of molecular modeling, mutagenesis, and X-ray crystallography. The structure of the extracellular region of CD40L was determined by protein crystallography, while the CD40 receptor was built using homology modeling based upon a novel alignment of the TNF receptor superfamily, and using the X-ray structure of the TNF receptor as a template. The model shows that the interface of the complex is composed of charged residues, with CD40L presenting basic side chains (K143, R203, R207), and CD40 presenting acidic side chains (D84, E114, E117). These residues were studied experimentally through site-directed mutagenesis, and also theoretically using electrostatic calculations with the program Delphi. The mutagenesis data explored the role of the charged residues in both CD40L and CD40 by switching to Ala (K143A, R203A, R207A of CD40L, and E74A, D84A, E114A, E117A of CD40), charge reversal (K143E, R203E, R207E of CD40L, and D84R, E114R, E117R of CD40), mutation to a polar residue (K143N, R207N, R207Q of CD40L, and D84N, E117N of CD40), and for the basic side chains in CD40L, isosteric substitution to a hydrophobic side chain (R203M, R207M). All the charge-reversal mutants and the majority of the Met and Ala substitutions led to loss of binding, suggesting that charged interactions stabilize the complex. This was supported by the Delphi calculations which confirmed that the CD40/CD40L residue pairs E74-R203, D84-R207, and E117-R207 had a net stabilizing effect on the complex. However, the substitution of hydrophilic side chains at several of the positions was tolerated, which suggests that although charged interactions stabilize the complex, charge per se is not crucial at all positions. Finally, we compared the electrostatic surface of TNF/TNFR with CD40L/CD40 and have identified a set of polar interactions surrounded by a wall of hydrophobic residues that appear to be similar but inverted between the two complexes.

Amino Acid Sequence↗
PubMed1998

Modularity in the TNF-receptor family.

Tumour necrosis factor (TNF) receptor family members regulate processes that range from cell proliferation to programmed cell death. The extracellular, ligand-binding domains of these proteins consist of small, cysteine-rich subdomains, first observed in the three-dimensional structures of the type I TNF receptor. A structure-based alignment of TNFR family members indicates that the extracellular domains are constructed primarily of two small polypeptide modules. These modules play distinctive structural roles in the architecture of the domains. Analogues of at least one of these modules can be found in the domains of other receptors and extracellular proteins. Variations in their sequence and order of assembly are expected to account for differences in shape, flexibility and ligand specificity.

Amino Acid Sequence↗
PubMed1998

Concanavalin A distorts the beta-GlcNAc-(1-->2)-Man linkage of beta-GlcNAc-(1-->2)-alpha-Man-(1-->3)-[beta-GlcNAc-(1-->2)-alpha-Man- (1-->6)]-Man upon binding.

Carbohydrate recognition by proteins is a key event in many biological processes. Concanavalin A is known to specifically recognize the pentasaccharide core (beta-GlcNAc-(1-->2)-alpha- Man-(1-->3)-[beta-GlcNAc-(1-->2)-alpha-Man-(1-->6)]-Man) of N-linked oligosaccharides with a Ka of 1.41 x 10(6 )M-1. We have determined the structure of concanavalin A bound to beta-GlcNAc-(1-->2)-alpha-Man-(1-->3)-[beta-GlcNAc-(1-->2)-alpha-Man- (1-->6)]-Man to 2.7A. In six of eight subunits there is clear density for all five sugar residues and a well ordered binding site. The pentasaccharide adopts the same conformation in all eight subunits. The binding site is a continuous extended cleft on the surface of the protein. Van der Waals interactions and hydrogen bonds anchor the carbohydrate to the protein. Both GlcNAc residues contact the protein. The GlcNAc on the 1-->6 arm of the pentasaccharide makes particularly extensive contacts and including two hydrogen bonds. The binding site of the 1-->3 arm GlcNAc is much less extensive. Oligosaccharide recognition by Con A occurs through specific protein carbohydrate interactions and does not require recruitment of adventitious water molecules. The beta-GlcNAc-(1-->2)-Man glycosidic linkage PSI torsion angle on the 1-->6 arm is rotated by over 50 degrees from that observed in solution. This rotation is coupled to disruption of interactions at the monosaccharide site. We suggest destabilization of the monosaccharide site and the conformational strain reduces the free energy liberated by additional interactions at the 1-->6 arm GlcNAc site.

Animals↗
PubMed1996

Structures of the extracellular domain of the type I tumor necrosis factor receptor.

BACKGROUND: Tumor necrosis factor (TNF) is a powerful cytokine that is involved in immune and pro-inflammatory responses. Two TNF receptors that belong to the cysteine-rich low affinity nerve growth factor receptor family (TNF-R1 and TNF-R2) are the sole mediators of TNF signalling. Signalling is thought to occur when a trimer of TNF binds to the extracellular domains of two or three receptor molecules, which permits aggregation and activation of the cytoplasmic domains. The complex is then internalized within an endocytic vesicle, whereupon it dissociates at low pH. Structure of the soluble extracellular domain of the receptor (sTNF-R1) both in the unliganded and TNF-bound state have previously been determined. In both instances, the fourth subdomain of the receptor was found to be partly disordered. In the unliganded state at pH 7.5, the extracellular domain forms two distinct types of dimer, parallel and antiparallel; the antiparallel dimer occludes the TNF-binding. RESULTS: We have determined the structure of sTNF-R1 in two crystal forms in high salt at pH 3.7. The orthorhombic crystals diffract to 1.85 ånd the entire polypeptide is well ordered. In contrast, the C-terminal 32 residues are disordered in the hexagonal crystals. In the orthorhombic form, these residues exhibit a topology and disulphide connectivity that differs from the other three cysteine-rich domains in the molecule. In both forms, the interface is considerably more extensive than that used in complex formation with LTalpha. This 'low pH' dimer is different from both of the dimers observed in crystals grown at pH 7.5. CONCLUSIONS: The occurrence of the antiparallel dimers in both low pH crystal forms suggest that they are not an artefact of crystal packing. Such dimers may form in the low pH environment of the endosome. Because the dimer contact surface occludes the TNF-binding site, formation of this dimer would dissociate the TNF-receptor complex within the endosome. Three of the four cysteine-rich domains of TNF-R1 are constructed from two distinct structural modules, termed A1 and B2. The fourth subdomain comprises an A1 module followed by an unusual C2 module. Although the orientation of these modules with respect to each other is sensitive to crystal packing, ligand binding, pH and ionic strength, the modules are structurally well conserved between and within the known sTNF-R1 structures.

Antigens, CD↗
PubMed1996

The crystal structure of a class II fructose-1,6-bisphosphate aldolase shows a novel binuclear metal-binding active site embedded in a familiar fold.

BACKGROUND: [corrected] Aldolases catalyze a variety of condensation and cleavage reactions, with exquisite control on the stereochemistry. These enzymes, therefore, are attractive catalysts for synthetic chemistry. There are two classes of aldolase: class I aldolases utilize Schiff base formation with an active-site lysine whilst class II enzymes require a divalent metal ion, in particular zinc. Fructose-1,6-bisphosphate aldolase (FBP-aldolase) is used in gluconeogenesis and glycolysis; the enzyme controls the condensation of dihydroxyacetone phosphate with glyceraldehyde-3-phosphate to yield fructose-1,6-bisphosphate. Structures are available for class I FBP-aldolases but there is a paucity of detail on the class II enzymes. Characterization is sought to enable a dissection of structure/activity relationships which may assist the construction of designed aldolases for use as biocatalysts in synthetic chemistry. RESULTS: The structure of the dimeric class II FBP-aldolase from Escherichia coli has been determined using data to 2.5 A resolution. The asymmetric unit is one subunit which presents a familiar fold, the (alpha/beta)8 barrel. The active centre, at the C-terminal end of the barrel, contains a novel bimetallic-binding site with two metal ions 6.2 A apart. One ion, the identity of which is not certain, is buried and may play a structural or activating role. The other metal ion is zinc and is positioned at the surface of the barrel to participate in catalysis. CONCLUSIONS: Comparison of the structure with a class II fuculose aldolase suggests that these enzymes may share a common mechanism. Nevertheless, the class II enzymes should be subdivided into two categories on consideration of subunit size and fold, quaternary structure and metal-ion binding sites.

Amino Acid Sequence↗