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G G Dodson

Publications and source records attributed to G G Dodson.

At least 19 recordsLinked to original sources
PubMed1998

Crystal structure of tryptophanase.

The X-ray structure of tryptophanase (Tnase) reveals the interactions responsible for binding of the pyridoxal 5'-phosphate (PLP) and atomic details of the K+ binding site essential for catalysis. The structure of holo Tnase from Proteus vulgaris (space group P2(1)2(1)2(1) with a = 115.0 A, b = 118.2 A, c = 153.7 A) has been determined at 2.1 A resolution by molecular replacement using tyrosine phenol-lyase (TPL) coordinates. The final model of Tnase, refined to an R-factor of 18.7%, (Rfree = 22.8%) suggests that the PLP-enzyme from observed in the structure is a ketoenamine. PLP is bound in a cleft formed by both the small and large domains of one subunit and the large domain of the adjacent subunit in the so-called "catalytic" dimer. The K+ cations are located on the interface of the subunits in the dimer. The structure of the catalytic dimer and mode of PLP binding in Tnase resemble those found in aspartate amino-transferase, TPL, omega-amino acid pyruvate aminotransferase, dialkylglycine decarboxylase (DGD), cystathionine beta-lyase and ornithine decarboxylase. No structural similarity has been detected between Tnase and the beta 2 dimer of tryptophan synthase which catalyses the same beta-replacement reaction. The single monovalent cation binding site of Tnase is similar to that of TPL, but differs from either of those in DGD.

Amino Acid Sequence↗
PubMed1997

The crystal structure of Citrobacter freundii tyrosine phenol-lyase complexed with 3-(4'-hydroxyphenyl)propionic acid, together with site-directed mutagenesis and kinetic analysis, demonstrates that arginine 381 is required for substrate specificity.

The X-ray structure of tyrosine phenol-lyase (TPL) complexed with a substrate analog, 3-(4'-hydroxyphenyl)propionic acid, shows that Arg 381 is located in the substrate binding site, with the side-chain NH1 4.1 A from the 4'-OH of the analog. The structure has been deduced at 2.5 A resolution using crystals that belong to the P2(1)2(1)2 space group with a = 135.07 A, b = 143.91 A, and c = 59.80 A. To evaluate the role of Arg 381 in TPL catalysis, we prepared mutant proteins replacing arginine with alanine (R381A), with isoleucine (R381I), and with valine (R381V). The beta-elimination activity of R381A TPL has been reduced by 10(-4)-fold compared to wild type, whereas R381I and R381V TPL exhibit no detectable beta-elimination activity with L-tyrosine as substrate. However, R381A, R381I, and R381V TPL react with S-(o-nitrophenyl)-L-cysteine, beta-chloro-L-alanine, O-benzoyl-L-serine, and S-methyl-L-cysteine and exhibit k(cat) and k(cat)/Km values comparable to those of wild-type TPL. Furthermore, the Ki values for competitive inhibition by L-tryptophan and L-phenylalanine are similar for wild-type, R381A, and R381I TPL. Rapid-scanning-stopped flow spectroscopic analyses also show that wild-type and mutant proteins can bind L-tyrosine and form quinonoid complexes with similar rate constants. The binding of 3-(4'-hydroxyphenyl)propionic acid to wild-type TPL decreases at high pH values with a pKa of 8.4 and is thus dependent on an acidic group, possibly Arg404, which forms an ion pair with the analog carboxylate, or the pyridoxal 5'-phosphate Schiff base. R381A TPL shows only a small decrease in k(cat)/Km for tyrosine at lower pH, in contrast to wild-type TPL, which shows two basic pKas with an average value of about 7.8. Thus, it is possible that Arg 381 is one of the catalytic bases previously observed in the pH dependence of k(cat)/Km of TPL with L-tyrosine [Kiick, D. M., & Phillips. R. S. (1988) Biochemistry 27, 7333-7338], and hence Arg 381 is at least partially responsible for the substrate specificity of TPL.

Arginine↗
PubMed1997

RNA cleavage without hydrolysis. Splitting the catalytic activities of binase with Asn101 and Thr101 mutations.

Members of the microbial guanyl-specific ribonuclease family catalyse the endonucleolytic cleavage of single-stranded RNA in a two-step reaction involving transesterification to form a 2',3'-cyclic phosphate and its subsequent hydrolysis to yield the respective 3'-phosphate. The extracellular ribonuclease from Bacillus intermedius (binase, RNase Bi) shares a common mechanism for RNA hydrolysis with mammalian RNases. Two catalytic residues in the active site of binase, Glu72 and His101, are thought to be involved in general acid-general base catalysis of RNA cleavage. Using site-directed mutagenesis, binase mutants were produced containing amino acid substitutions H101N and H101T and their catalytic properties towards RNA, poly(I), poly(A), GpC and guanosine 2',3'-cyclic phosphate (cGMP) substrates were studied. The engineered mutant proteins are active in the transesterification step which produces the 2',3'-cyclic phosphate species but they have lost the ability to catalyse hydrolysis of the cyclic phosphate to give the 3' monophosphate product.

Asparagine↗
PubMed1997

A model of insulin fibrils derived from the x-ray crystal structure of a monomeric insulin (despentapeptide insulin).

The crystal structure of despentapeptide insulin, a monomeric insulin, has been refined at 1.3 A spacing and subsequently used to predict and model the organization in the insulin fibril. The model makes use of the contacts in the densely packed despentapeptide insulin crystal, and takes into account other experimental evidence, including binding studies with Congo red. The dimensions of this model fibril correspond well with those measured experimentally, and the monomer-monomer contacts within the fibril are in accordance with the known physical chemistry of insulin fibrils. Using this model, it may be possible to predict mutations in insulin that might alleviate problems associated with fibril formation during insulin therapy.

Computer Simulation↗
PubMed1996

The binding of carbon monoxide and nitric oxide to leghaemoglobin in comparison with other haemoglobins.

Haemoglobins have the ability to discriminate between oxygen and other diatomic molecules. To further understanding of this process the X-ray crystal structures of carbonmonoxy and nitrosyl-leghaemoglobin have been determined at 1.8 A resolution. The ligand geometry is discussed in detail and the controversial issue of bent versus linear carbon monoxide binding is addressed. The bond angle of 160 degrees for CO-leghaemoglobin is in conflict with recent spectroscopy results on myoglobin but is consistent with angles obtained for myoglobin X-ray crystal structures. In contrast to the numerous carbon monoxide studies, very little stereochemical information is available for the nitric oxide adduct of haemoglobin. This is provided by the X-ray structure of NO-leghaemoglobin, which conforms to expected geometry with an Fe-NO angle of 147 degrees and a lengthened iron-proximal histidine bond. Thus crystallographic evidence is given for the predicted weakening of this bond on the binding of nitric oxide.

Binding Sites↗
PubMed1996

Site-directed mutagenesis of the base recognition loop of ribonuclease from Bacillus intermedius (binase).

Members of the microbial guanyl-specific ribonuclease family show a high level of structural homology. The structural basis for guanyl base binding by microbial ribonucleases has been established for all members of the family and the existence of a guanine recognition loop was shown. However, bacillar RNases such as binase and barnase show far less specificity towards the guanyl base in hydrolysing oligonucleotides composed of more than 4 or 5 nucleotides. Using site-directed mutagenesis we introduced a number of amino acid substitutions into the base recognition loop of binase. The donor sequence originated from the guanyl specific ribonuclease Sa. Two single, two double and one triple (entire loop substitution) mutants were constructed and overproduced in E. coli. The kinetic properties of the mutant variants are different from the wild-type protein. Amino acid substitutions R61V, G60S, S56Q/R61V, G60S/R61V show 3-fold, 7-fold, 4-fold and 12-fold increased guanyl specificity respectively. However, all mutants retain the ability to catalyse the hydrolysis of a poly(A) substrate.

Amino Acid Sequence↗
PubMed1996

Circular assemblies.

During 1994 and 1995, the structures of the serum amyloid P component, the bacterial chaperonin GroEL, the 20S proteasome, the bacterial light-harvesting complexes and the tryptophan operon RNA-binding attenuation protein have been determined. These structures all form circular assemblies in which the individual subunits are related by rotational symmetry. In most cases the circular organization generates a new biophysical property and a specific biological function which have presumably been selected by evolution.

Bacterial Proteins↗
PubMed1995

X-ray crystallographic studies on hexameric insulins in the presence of helix-stabilizing agents, thiocyanate, methylparaben, and phenol.

Three X-ray crystallographic studies have been carried out on pig insulin in the presence of three ligands, thiocyanate, methylparaben (methyl p-hydroxybenzoate), and phenol. In each case, rhombohedral crystals were obtained, which diffracted to 1.8, 1.9, and 2.3 A, respectively. Each crystal structure was very similar to that of 4-zinc pig insulin, which was used as a starting model for PROLSQ refinement (Collaborative Computational Project, Number 4, 1994). The R factors for the refined structures of thiocyanate insulin, methylparaben insulin, and phenol insulin were 19.6, 18.4, and 19.1, respectively. Each crystal structure consists of T3R3f insulin hexamers with two zinc ions per hexamer. In the R3f trimer of the thiocyanate insulin hexamer, one thiocyanate ion is coordinated to the zinc on the hexamer 3-fold axis, but there is no evidence of zinc ion binding in the off-axis zinc ion sites seen in the 4-zinc pig insulin structure. In the methylparaben insulin and phenol insulin hexamers, the phenolic ligands are bound at the dimer-dimer interfaces in the R3f trimers in a manner similar to that of phenol in R6 phenol insulin. The binding of methylparaben appears to make the hexamer more compact by drawing the A and the B chains closer together in the binding site. In all three structures presented herein, the conformations of the first three residues of the B chain in the R3f trimer are extended rather than alpha-helical, as is seen in R6 phenol insulin. The energetics of ligand binding in the insulin hexamer are discussed.

Animals↗
PubMed1995

The structure of deoxy- and oxy-leghaemoglobin from lupin.

The leghaemoglobins have oxygen affinities 11 to 24 times higher than that of sperm whale myoglobin, due mainly to higher rates of association. To find out why, we have determined the structures of deoxy- and oxy-leghaemoglobin II of the lupin at 1.7 A resolution. Results confirm the general features found in previous X-ray analyses of this protein. The unique feature that has now emerged is the rotational freedom of the proximal histidine. In deoxy-leghaemoglobin the imidazole oscillates between two alternative orientations, eclipsing either the lines N1-N3 or N2-N4 of the porphyrin; in oxy-leghaemoglobin it is fixed in a staggered orientation. The iron atom moves from a position 0.30 A from the plane of the pyrrole nitrogen atoms in deoxy- to a position in the plane in oxy-leghaemoglobin while the Fe- bond distance remains constant at 2.02 A. The Fe-O-O angle is 152 degrees, as in human haemoglobin. The oxygen is hydrogen-bonded to the distal histidine at N epsilon 2-O1 and N epsilon 2-O2 distance of 2.95 A and 2.68 A, respectively. The porphyrin is ruffled equally in deoxy- and oxy-leghaemoglobins, due to rotations of the pyrrols about the N-Fe-N bonds, causing the methine bridges to deviate by up to 0.32 A from the mean porphyrin plane. The only feature capable of accounting for the high on-rate of the reaction with oxygen are the mobilities of the proximal histidine and distal histidine residues in deoxy-leghaemoglobin. The eclipsed positions of the proximal histidine in deoxy-leghaemoglobin maximize steric hindrance with the porphyrin nitrogen atoms and minimize pi-->p electron donation, while its staggered position in oxy-leghaemoglobin reverses both these effects. Together with the oscillation of the imidazole between the two orientations, these two factors may reduce the activation energy for the reaction of leghaemoglobin with oxygen. The distal histidine is in a fixed position in the haem pocket in the crystal, but must be swinging in and out of the pocket at a high rate in solution to allow the oxygen to enter.

Crystallography, X-Ray↗
PubMed1995

Structure of a 14-3-3 protein and implications for coordination of multiple signalling pathways.

A broad range of organisms and tissues contain 14-3-3 proteins, which have been associated with many diverse functions including critical roles in signal transduction pathways, exocytosis and cell cycle regulation. We report here the crystal structure of the human T-cell 14-3-3 isoform (tau) dimer at 2.6 A resolution. Each monomer (Mr 28K) is composed of an unusual arrangement of nine antiparallel alpha-helices organized as two structural domains. The dimer creates a large, negatively charged channel approximately 35 A broad, 35 A wide and 20 A deep. Overall, invariant residues line the interior of this channel whereas the more variable residues are distributed on the outer surface. At the base of this channel is a 16-residue segment of 14-3-3 which has been implicated in the binding of 14-3-3 to protein kinase C.

14-3-3 Proteins↗
PubMed1995

The structure of trp RNA-binding attenuation protein.

The crystal structure of the trp RNA-binding attenuation protein of Bacclius subtilis solved at 1.8 A resolution reveals a novel structural arrangement in which the eleven subunits are stabilized through eleven intersubunit beta-sheets to form a beta-wheel with a large central hole. The nature of the binding of L-tryptophan in clefts between adjacent beta-sheets in the beta-wheel suggests that this binding induces conformational changes in the flexible residues 25-33 and 49-52. It is argued that upon binding, the messenger RNA target forms a matching circle in which eleven U/GAG repeats are bound to the surface of the protein ondecamer modified by the binding of L-tryptophan.

Amino Acid Sequence↗
PubMed1994

Crystal structure of an extracellular fragment of the rat CD4 receptor containing domains 3 and 4.

BACKGROUND: CD4 is a transmembrane protein on the surface of T lymphocytes that interacts with MHC class II proteins at the surface of accessory cells, and is involved in the triggering of the lymphocytes by foreign antigens. It is also the major receptor for the human immunodeficiency virus. The extracellular portion of CD4 was predicted to contain four immunoglobulin superfamily domains and this has been confirmed by X-ray crystallography, but no detailed structure of domains 3 and 4 has been available. RESULTS: We now report the expression of a form of rat CD4 containing only domains 3 and 4, its crystallization, and the refinement and analysis of its structure by X-ray crystallography with 2.6 A spacing data. Both domains show variations in core residues when compared with immunoglobulin domains. Features of the structure are discussed with respect to the structure of the complete extracellular part of CD4 and its function. CONCLUSIONS: Domains 3 and 4 of CD4 show considerable similarity to domains 1 and 2, although there is a 25 degrees rotation in the relative positions of the domains with respect to one another. The absence of the disulphide bond in domain 3 is associated with an alteration in the packing of the beta-sheets, which may be important for interactions with domain 2 in the overall receptor structure. The location of N-linked glycosylation on one face of domain 3 appears to preclude the dimerization that is observed in antibodies.

Amino Acid Sequence↗
PubMed1994

The structural basis of sequence-independent peptide binding by OppA protein.

Specific protein-ligand interactions are critical for cellular function, and most proteins select their partners with sharp discrimination. However, the oligopeptide-binding protein of Salmonella typhimurium (OppA) binds peptides of two to five amino acid residues without regard to sequence. The crystal structure of OppA reveals a three-domain organization, unlike other periplasmic binding proteins. In OppA-peptide complexes, the ligands are completely enclosed in the protein interior, a mode of binding that normally imposes tight specificity. The protein fulfills the hydrogen bonding and electrostatic potential of the ligand main chain and accommodates the peptide side chains in voluminous hydrated cavities.

Amino Acid Sequence↗
PubMed1994

An unusual buried polar cluster in a family of fungal lipases.

The stability of globular proteins arises largely from the burial of non-polar amino acids in their interior. These residues are efficiently packed to eliminate energetically unfavorable cavities. Contrary to these observations, high resolution X-ray crystallographic analyses of four homologous lipases from filamentous fungi reveal an alpha/beta fold which contains a buried conserved constellation of charged and polar side chains with associated cavities containing ordered water molecules. It is possible that this structural arrangement plays an important role in interfacial catalysis.

Amino Acid Sequence↗
PubMed1994

Epitope analysis of human insulin and intact proinsulin.

Residues belonging to epitopes on human insulin that were recognized by a panel of three monoclonal antibodies were located using mutated insulins and insulins from a number of different animal species. Epitopes on human proinsulin recognized by two monoclonal antibodies were also identified using partially processed proinsulin species. Epitopes were located on the C-A and B-C junctions of proinsulin and on the N-termini of the A- and B-chains and the central region of the B-chain of human insulin. Antibodies that bound proinsulin were found to induce conformational changes in the prohormone. The presence of a well-defined interaction between the C-peptide portion and the N-terminus of the A-chain of the insulin moiety of intact proinsulin has also been demonstrated. The relevance of these studies to the development of two-site assays for the measurement of partially processed proinsulin species in human sera is also discussed.

Animals↗
PubMed1994

Probing the nature of substrate binding in Humicola lanuginosa lipase through X-ray crystallography and intuitive modelling.

The catalytic triad of the neutral lipase from Humicola lanuginosa is buried by a short helix under aqueous conditions rendering the enzyme inactive. Upon adsorption to a lipid substrate interface this helix is displaced, thereby exposing the active site (interfacial activation). By covalently linking inhibitors to the active serine, it is possible to crystallize the enzyme in an interfacially activated state. Two such structures are reported here which mimic the tetrahedral transition states of lipolysis. To date, no crystal structures of a lipase--triglyceride complex exist for this enzyme. Therefore, possible interactions between this lipase and its substrate have been analysed through molecular modelling.

Binding Sites↗
PubMed1993

Structure and function of endoglucanase V.

Cellulose is the major polysaccharide component of plant cell walls and is the most abundant organic compound on the planet. A number of bacterial and fungal organisms can use cellulose as a food source, possessing cellulases (cellobiohydrolases and endoglucanases) that can catalyse the hydrolysis of the beta-(1,4) glycosidic bonds. They can be classified into seven distinct families. The three-dimensional structures of members of two of these families are known. Here we report the structure of a third cellulase, endoglucanase V, whose sequence is not represented in any of the above families. The enzyme is structurally distinct from the previously determined cellulases but is similar to a recently characterized plant defence protein. The active site region resembles that of lysozyme, despite the lack of structural similarity between these two enzymes.

Binding Sites↗