Biomedical subjects

M N James

Publications and source records attributed to M N James.

At least 73 records · Page 4Linked to original sources

PubMed1994

The crystal structure of succinyl-CoA synthetase from Escherichia coli at 2.5-A resolution.

The x-ray crystal structure of succinyl-CoA synthetase (SCS) from Escherichia coli has been determined by the method of multiple isomorphous replacement to a resolution of 2.5 A. Crystals of SCS are tetragonal with a space group of P4(3)22 and unit cell dimensions of a = b = 98.47 A and c = 400.6 A. One molecule of SCS (142 kDa) is contained in the asymmetric unit. The current model has been refined to a conventional R factor of 21.6% with root mean square deviations from ideal stereochemistry of 0.022 A for bond lengths and 3.25 degrees for bond angles. The quaternary organization of the E. coli enzyme is an alpha 2 beta 2 heterotetramer. In this tetramer, the alpha-subunits interact only with the beta-subunits, whereas the beta-subunits interact to form the dimer of alpha beta-dimers. The two active site pockets are located at regions of contact between alpha- and beta-subunits. One molecule of coenzyme A is bound to each alpha-subunit at a typical nucleotide-binding motif, and His-246 of each alpha-subunit is phosphorylated. This phosphohistidine, a catalytic intermediate, is stabilized by two helix dipoles (the "power" helices), one from each of the two subunit types. A short segment of the beta-subunit from one alpha beta-dimer is in close proximity to the CoA-binding site of the other alpha beta-dimer, providing a possible rationale for the overall tetrameric structure.

Amino Acid Sequence↗

PubMed1994

Refined 1.7 A X-ray crystallographic structure of P-30 protein, an amphibian ribonuclease with anti-tumor activity.

The X-ray crystallographic structure of P-30 protein (Onconase) has been solved by multiple isomorphous replacement and the structure has been refined at 1.7 A resolution to a conventional R-factor of 0.178. The molecular model comprises all 826 non-hydrogen protein atoms, 96 solvent molecules and a sulfate anion that is bound at the active site. The molecular structure is similar to that of ribonuclease A. The active site cleft is located at the junction of two three-stranded beta-sheets and the N-terminal helix. A sulfate anion is non-covalently bound by Lys9, His10, His97, Phe98 and an intermolecular contact involving Lys55' from a neighboring molecule. The N-terminal pyroglutamyl (Pyr) residue is part of the active site and its O epsilon 1 atom forms a hydrogen bond with the Lys9 N zeta. The previously constructed comparative molecular model of P-30 based on ribonuclease A correctly predicted the overall fold of P-30 and the conformation of its active site residues. The model failed to predict the conformation of Pyr1 and the conformation of the two loops following helix alpha 3 and strand beta 3.

Animals↗

PubMed1994

Refined crystal structure of rat parvalbumin, a mammalian alpha-lineage parvalbumin, at 2.0 A resolution.

We present here the X-ray crystal structure of the rat alpha-parvalbumin from fast twitch muscle. This protein (M(r) 11.8 kDa) crystallizes in space group P2(1)2(1)2(1) with unit cell dimensions of a = 34.3 A, b = 55.0 A, c = 156.1 A and three molecules in the asymmetric unit. The protein structure was solved by the molecular replacement method and has been refined to a crystallographic R-factor [formula: see text] of 0.181 for all reflections with I/sigma(I) > or = 2 (I = intensity) between 8.0 and 2.0 A resolution. The molecules located most easily in the molecular replacement rotation function had lower overall thermal motion parameters and higher numbers of intermolecular crystal packing contacts. The overall fold of the polypeptide chain for the rat alpha-parvalbumin is similar to other known parvalbumin structures (root-mean-square deviations in alpha-carbon atom positions range from 0.60 to 0.87 A). There are two Ca(2+)-binding sites in parvalbumins, and there is some evidence for a third ion-binding site, adjacent to the CD site, in the rat species. The level of structural variability among the best-ordered regions of the three independent rat alpha-parvalbumin molecules in the crystallographic asymmetric unit is two to three times higher than the mean coordinate error (0.10 A), indicating flexibility in the molecule. Sequence differences between alpha and beta-lineage parvalbumins result in repacking of the hydrophobic core and some shifts in the protein backbone. The shifts are localized, however, and entire helices do not shift as rigid units.

Animals↗

PubMed1994

Common structural features of the luxF protein and the subunits of bacterial luciferase: evidence for a (beta alpha)8 fold in luciferase.

The amino acid sequence identity and potential structural similarity between the subunits of bacterial luciferase and the recently determined structure of the luxF molecule are examined. The unique beta/alpha barrel fold found in luxF appears to be conserved in part in the luciferase subunits. From secondary structural predictions of both luciferase subunits, and from structural comparisons between the protein product of the luxF gene, NFP, and glycolate oxidase, we propose that it is feasible for both luciferase subunits to adopt a (beta alpha)8 barrel fold with at least 2 excursions from the (beta alpha)8 topology. Amino acids conserved between NFP and the luciferase subunits cluster together in 3 distinct "pockets" of NFP, which are located at hydrophobic interfaces between the beta-strands and alpha-helices. Several tight turns joining the C-termini of beta-strands and the N-termini of alpha-helices are found as key components of these conserved regions. Helix start and end points are easily demarcated in the luciferase subunit protein sequences; the N-cap residues are the most strongly conserved structural features. A partial model of the luciferase beta subunit from Photobacterium leiognathi has been built based on our crystallographically determined structure of luxF at 1.6 A resolution.

Alcohol Oxidoreductases↗

PubMed1994

Crystal structure of the holotoxin from Shigella dysenteriae at 2.5 A resolution.

Shigella dysenteriae is the pathogen responsible for the severe form of dysentery in humans. It produces Shiga toxin, the prototype of a family of closely related bacterial protein toxins. We have determined the structure of the holotoxin, an AB5 hexamer, by X-ray crystallography. The five B subunits form a pentameric ring, encircling a helix at the carboxy terminus of the A subunit. The A subunit interacts with the B pentamer via this C-terminal helix and a four-stranded mixed beta-sheet. The fold of the rest of the A subunit is similar to that of the A chain of the plant toxin ricin; both are N-glycosidases. However, the active site in the bacterial holotoxin is blocked by a segment of polypeptide chain. These residues of the A subunit would be released as part of the activation mechanism of the toxin.

Amino Acid Sequence↗

PubMed1994

Conversion of the substrate specificity of mouse proteinase granzyme B.

Mouse granzyme B is the prototypic member of a subfamily of serine proteinases expressed in cytolytic lymphocytes. Molecular modelling of granzyme B indicated that the side chain of Arg 208 partially fills the specificity pocket, thus predicting the preference of this enzyme for substrates containing acidic side chains, a feature unique among eukaryotic serine proteinases. Replacement of Arg 208 with glycine results in an enzyme lacking this activity, but which is able to hydrolyze hydrophobic substrates. These results demonstrate unequivocally that the substrate preference of granzyme B is determined by a positive charge in the specificity pocket and also represent one of the few examples of rational and efficient alteration of serine proteinase substrate-specificity following a single amino acid substitution.

Amino Acid Sequence↗

PubMed1993

Hepatitis A virus 3C proteinase: some properties, crystallization and preliminary crystallographic characterization.

Several isoforms of the wild-type and three mutant hepatitis A virus (HAV) 3C proteinases have been isolated and characterized. The active site cysteine residue (residue 172) was found to be responsible for the formation of some of these isoforms. The double mutant C24S/C172A of the HAV 3C proteinase, in which both cysteine residues have been replaced by site-directed mutagenesis, was crystallized. The crystals belong to the hexagonal space group P6(1)22 (or its enantiomorph, P6(5)22) with unit cell dimensions a = b = 65.2 A, c = 246.1 A and diffract X-rays to 2.3 A resolution.

3C Viral Proteases↗

PubMed1993

Purification and crystallization of Shiga toxin from Shigella dysenteriae.

The protein toxin produced by Shigella dysenteriae consists of one enzymatically active A subunit of 293 amino acid residues and five B subunits of 69 amino acid residues that are involved with cell attachment. The holotoxin has been purified by blue Sepharose and chromatofocusing column chromatography. Two crystal forms of purified holotoxin have been grown by vapor diffusion. One grows as fine needles, hexagonal in cross-section, which do not diffract well enough to characterize crystallographically. The second grows as thin plates that diffract to at least 3 A resolution. Their space group is P2(1)2(1)2(1) with unit cell dimensions of a = 132.0 A, b = 146.0 A and c = 82.5 A. The asymmetric unit of the crystals is likely to contain two AB5 units.

Bacterial Toxins↗

PubMed1993

Crystal structure of a flavoprotein related to the subunits of bacterial luciferase.

The molecular structure of the luxF protein from the bioluminescent bacterium Photobacterium leiognathi has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 17.8% at 2.3 A resolution. The 228 amino acid polypeptide exists as a symmetrical homodimer and 33% of the monomer's solvent-accessible surface area is buried upon dimerization. The monomer displays a novel fold that contains a central seven-stranded beta-barrel. The solvent-exposed surface of the monomer is covered by seven alpha-helices, whereas the dimer interface is primarily a flat surface composed of beta-strands. The protein monomer binds two molecules of flavin mononucleotide, each of which has C6 of the flavin isoalloxazine moiety covalently attached to the C3' carbon atom of myristic acid. Both myristyl groups of these adducts are buried within the hydrophobic core of the protein. One of the cofactors contributes to interactions at the dimer interface. The luxF protein displays considerable amino acid sequence homology with both alpha- and beta-subunits of bacterial luciferase, especially the beta-subunit. Conserved amino acid residues shared between luxF and the luciferase subunits cluster predominantly in two distinct regions of the luxF protein molecule. These homologous regions in the luciferase subunits probably share a three-dimensional fold similar to that of the luxF protein.

Amino Acid Sequence↗

PubMed1992

Molecular structure of the acyl-enzyme intermediate in beta-lactam hydrolysis at 1.7 A resolution.

The X-ray crystal structure of the molecular complex of penicillin G with a deacylation-defective mutant of the RTEM-1 beta-lactamase from Escherichia coli shows how these antibiotics are recognized and destroyed. Penicillin G is covalently bound to Ser 70 0 gamma as an acyl-enzyme intermediate. The deduced catalytic mechanism uses Ser 70 0 gamma as the attacking nucleophile during acylation. Lys 73 N zeta acts as a general base in abstracting a proton from Ser 70 and transferring it to the thiazolidine ring nitrogen atom via Ser 130 0 gamma. Deacylation is accomplished by nucleophilic attack on the penicilloyl carbonyl carbon by a water molecule assisted by the general base, Glu 166.

Acylation↗

PubMed1992

Crystallization of human beta-hexosaminidase B.

beta-Hexosaminidase is a lysosomal hydrolase that is important in the metabolism of sphingoglycolipids. beta-Hexosaminidase B and beta-hexosaminidase A are the major isozymes in normal human tissue. beta-Hexosaminidase B is a homodimer of beta subunits, and beta-hexosaminidase A is a heterodimer composed of an alpha and a beta subunit. Crystals of beta-hexosaminidase B (M(r) 112,000) have been grown using the handling drop technique. They are elongated hexagonal prisms with maximum dimensions of 0.2 mm x 0.2 mm x 0.7 mm. The space group is P6(1)22 (or enantiomorph); the unit cell dimensions are a = b = 114.2 A, c = 402.2 A, alpha = beta = 90 degrees, gamma = 120 degrees. The molecular mass and cell dimensions suggest that there is one dimer per asymmetric unit. Crystals diffract to 3.2 A resolution.

Crystallization↗

PubMed1992

Crystallographic analysis of transition-state mimics bound to penicillopepsin: phosphorus-containing peptide analogues.

The molecular structures of three phosphorus-based peptide inhibitors of aspartyl proteinases complexed with penicillopepsin [1, Iva-L-Val-L-Val-StaPOEt [Iva = isovaleryl, StaP = the phosphinic acid analogue of statine [(S)-4-amino-(S)-3-hydroxy-6-methylheptanoic acid] (IvaVVStaPOEt)]; 2, Iva-L-Val-L-Val-L-LeuP-(O)Phe-OMe [LeuP = the phosphinic acid analogue of L-leucine; (O)Phe = L-3-phenyllactic acid; OMe = methyl ester] [Iva VVLP(O)FOMe]; and 3, Cbz-L-Ala-L-Ala-L-LeuP-(O)-Phe-OMe (Cbz = benzyloxycarbonyl) [CbzAALP(O)FOMe]] have been determined by X-ray crystallography and refined to crystallographic agreement factors, R ( = sigma parallel to F0 magnitude of - Fc parallel to/sigma magnitude of F0), of 0.132, 0.131, and 0.134, respectively. These inhibitors were designed to be structural mimics of the tetrahederal transition-state intermediate encountered during aspartic proteinase catalysis. They are potent inhibitors of penicillopepsin with Ki values of 1, 22 nM; 2, 2.8 nM; and 3, 1600 nM, respectively [Bartlett, P. A., Hanson, J. E., & Giannousis, P. P. (1990) J. Org. Chem. 55, 6268-6274]. All three of these phosphorus-based inhibitors bind virtually identically in the active site of penicillopepsin in a manner that closely approximates that expected for the transition state [James, M. N. G., Sielecki, A.R., Hayakawa, K., & Gelb, M. H. (1992) Biochemistry 31, 3872-3886]. The pro-S oxygen atom of the two phosphonate inhibitors and of the phosphinate group of the StaP inhibitor make very short contact distances (approximately 2.4 A) to the carboxyl oxygen atom, O delta 1, of Asp33 on penicillopepsin. We have interpreted this distance and the stereochemical environment of the carboxyl and phosphonate groups in terms of a hydrogen bond that most probably has a symmetric single-well potential energy function. The pro-R oxygen atom is the recipient of a hydrogen bond from the carboxyl group of Asp213. Thus, we are able to assign a neutral status to Asp213 and a partially negatively charged status to Asp33 with reasonable confidence. Similar very short hydrogen bonds involving the active site glutamic acid residues of thermolysin and carboxypeptidase A and the pro-R oxygen of bound phosphonate inhibitors have been reported [Holden, H. M., Tronrud, D. E., Monzingo, A. F., Weaver, L. H., & Matthews, B. W. (1987) Biochemistry 26, 8542-8553; Kim, H., & Lipscomb, W. N. (1991) Biochemistry 30, 8171-8180].(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1992

Separation of porcine pepsinogen A and progastricsin. Sequencing of the first 73 amino acid residues in progastricsin.

Porcine pepsinogen A (EC 3.4.23.1) and progastricsin (EC 3.4.23.3) have been separated by chromatography on DEAE-cellulose followed by chromatography on DEAE-Sepharose. Agar gel electrophoresis at pH 6.0 showed the presence of three components of pepsinogen A and two of progastricsin. During activation at pH 2 a segment of 43 amino acid residues (the prosegment peptide) is cleaved from the N-terminus of progastricsin. The sequence of this was determined; in addition, the first 30 residues of gastricsin were sequenced. The sequence of the first 73 amino acid residues of progastricsin shows an overall identity with progastricsins from man, monkey and rat of 67%. The overall identity with other zymogens for gastric proteinases is 27%. The highly conserved Lys36p (pig pepsinogen A numbering) is changed to Arg in porcine progastricsin.

Amino Acid Sequence↗

PubMed1992

Crystallographic analysis of transition state mimics bound to penicillopepsin: difluorostatine- and difluorostatone-containing peptides.

Difluorostatine- and difluorostatone-containing peptides have been evaluated as potent inhibitors of penicillopepsin, a member of the aspartic proteinase family of enzymes. Isovaleryl-Val-Val-StaF2NHCH3 [StaF2 = (S)-4-amino-2,2-difluoro-(R)-3-hydroxy-6-methylheptanoic acid] and isovaleryl-Val-Val-StoF2NHCH3 [StoF2 = (S)-4-amino-2,2-difluoro-3-oxo-6-methylheptanoic acid] have measured Ki's of 10 x 10(-9) and 1 x 10(-9) M, respectively, with this fungal proteinase. The StoF2-containing peptide binds 32-fold more tightly to the enzyme than the analogous peptide containing the non-fluorinated statine ethyl ester. Each compound was cocrystallized with penicillopepsin, intensity data were collected to 1.8-A resolution, and the atomic coordinates were refined to an R factor [formula: see text] of 0.131 for both complexes. The inhibitors bind in the active site of penicillopepsin in much the same fashion as do other statine-containing inhibitors of penicillopepsin analyzed earlier [James, M. N. G., Sielecki, A. Salituro, F., Rich, D. H., & Hofmann, T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6137-6141; James, M.N.G., Sielecki, A., & Hofmann, T. (1985) in Aspartic Proteinases and their Inhibitors (Kosta, V., Ed.) pp 163-177, Walter deGruyter, Berlin]. The (R)-3-hydroxyl group in StaF2 binds between the active site carboxyl groups of Asp33 and Asp213, making hydrogen-bonding contacts to each one. The ketone functional group of the StoF2 inhibitor is bound as a hydrated species, with the gem-diol situated between the two aspartic acid carboxyl groups in a manner similar to that predicted for the tetrahedral intermediate expected during the catalytic hydrolysis of a peptide bond [James, M. N. G., & Sielecki, A. (1985) Biochemistry 24, 3701-3713]. One hydrogen-bonding interaction from the "outer" hydroxyl group is made to O delta 1 of Asp33, and the "inner" hydroxyl group forms two hydrogen-bonding contacts, one to each of the carboxyl groups of Asp33 (O delta 2) and Asp213 (O delta 2). The only structural difference between the StaF2 and StoF2 inhibitors that accounts for the factor of 10 in their Ki's is the additional (R)-3-OH group on the tetrahedral sp3 carbon atom of the hydrated StoF2 inhibitor. The intermolecular interactions involving the fluorine atoms of each inhibitor are normal van der Waals contacts to one of the carboxyl oxygen atoms of Asp213 (F2-O delta 2 Asp213, 2.9 A). The observed stereochemistry of the bound StoF2 group in the active site of penicillopepsin has stimulated our reappraisal of the catalytic pathway for the aspartic proteinases.(ABSTRACT TRUNCATED AT 400 WORDS)

Aspartic Acid Endopeptidases↗

PubMed1992

Crystallization of Photobacterium leiognathi non-fluorescent flavoprotein, an unusual flavoprotein with limited sequence identity to bacterial luciferase.

Single crystals of the non-fluorescent flavoprotein (NFP) purified from Photobacterium leiognathi strain S1 have been grown from ammonium sulphate solutions using the hanging drop vapour diffusion technique. The crystals grow as thin (0.06 mm) plates and belong to the orthorhombic space group C222(1): a = 57.06(3) A, b = 92.41(6) A, c = 99.52(6) A. There is one NFP monomer per asymmetric unit and crystals diffract to 2.2 A spacings on film. A complete native data set to 2.5 A resolution has been collected on a San Diego Multiwire Detector system at the University of Alberta and a heavy-atom derivative search is presently in progress.

Bacterial Proteins↗

PubMed1992

Crystallization and preliminary X-ray diffraction studies of two mutants of lactate dehydrogenase from Bacillus stearothermophilus.

Bacillus stearothermophilus lactate dehydrogenase, one of the most thermostable bacterial enzymes known, has had its three-dimensional structure solved, the gene coding for it has been cloned, and the protein can be readily overexpressed. Two mutants of the enzyme have been prepared. In one, Arg171 was changed to Trp (R171W) and Gln102 was changed to Arg (Q102R). In the other, the mutation Q102R was maintained, but Arg171 was changed to Tyr (R171Y). In addition, an inadvertent C97G mutant was present. Both mutants have been crystallized by the hanging drop vapor diffusion method at room temperature. Bipyrimidal crystals have been obtained against (NH4)2SO4 in 50 mM piperazine HCl buffer. The crystals belong to space group P6(2)22 (P6(4)22) (whereas the native enzyme, the structure of which has been solved by Piontek et al., Proteins 7:74-92, 1990) crystallized in the space group P6(1)) with a = 102.3 A, c = 168.6 A for the R171W, Q102R, C97G triple mutant, and a = 98.2 A; c = 162.1 A for the R171Y, Q102R, C97G mutant. These crystal forms appear to contain one-quarter of a tetramer (M(r) 135,000) in the asymmetric unit and have VM values of 3.8 and 3.3 A3/dalton, respectively). The R171W mutant diffracts to 2.5 A and the R171 Y mutant to approximately 3.5 A.

Bacterial Proteins↗

PubMed1992

Comparative molecular modeling and crystallization of P-30 protein: a novel antitumor protein of Rana pipiens oocytes and early embryos.

The P-30 protein (Onconase) of Rana pipiens oocytes and early embryos is homologous to members of the pancreatic ribonuclease superfamily and exhibits an antitumor activity in vitro and in vivo. It appears that the ribonucleolytic activity of P-30 protein may be required for its antitumor effects. A comparative molecular model of P-30 protein has been constructed based upon the known three-dimensional structure of bovine pancreatic RNase A in order to provide structural information. Functionally, these enzymes hydrolyze oligoribonucleotides to pyrimidine-3'-phosphate monoesters and 5'-OH ribonucleotides. In the modeling procedure, automated sequence alignments were revised based upon the inspection of the RNase A structure before the amino acids of the P-30 protein were assigned the coordinates of the RNase A template. The inevitable intermolecular steric clashes that result were relieved on an interactive graphics device through the adjustment of side chain torsion angles. This process was followed by energy minimization of the model, which served to optimize stereochemical geometry and to relieve any remaining unacceptably close contacts. The resulting model retains the essential features of RNase A as sequence insertions and deletions are almost exclusively found in exposed surface loops. The all atom superposition of active site residues of the P-30 protein model and an identically minimized RNase A structure has a root mean square deviation of 0.52 A. Though tentative, the model is consistent with a pyrimidine specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

Comparative NMR studies on cardiac troponin C and a mutant incapable of binding calcium at site II.

One- and two-dimensional NMR techniques were used to study both the influence of mutations on the structure of recombinant normal cardiac troponin C (cTnC3) and the conformational changes induced by Ca2+ binding to site II, the site responsible for triggering muscle contraction. Spin systems of the nine Phe and three Tyr residues were elucidated from DQF-COSY and NOESY spectra. Comparison of the pattern of NOE connectivities obtained from a NOESY spectrum of cTnC3 with a model of cTnC based on the crystal structure of skeletal TnC permitted sequence-specific assignment of all three Tyr residues, as well as Phe-101 and Phe-153. NOESY spectra and calcium titrations of cTnC3 monitoring the aromatic region of the 1H NMR spectrum permitted localization of six of the nine Phe residues to either the N- or C-terminal domain of cTnC3. Analysis of the downfield-shifted C alpha H resonances permitted sequence-specific assignment of those residues involved in the beta-strand structures which are part of the Ca(2+)-binding loops in both the N- and C-terminal domains of cTnC3. The short beta-strands in the N-terminal domain of cTnC3 were found to be present and in close proximity even in the absence of Ca2+ bound at site II. Using these assignments, we have examined the effects of mutating Asp-65 to Ala, CBM-IIA, a functionally inactive mutant which is incapable of binding Ca2+ at site II [Putkey, J.A., Sweeney, H. L., & Campbell, S. T. (1989) J. Biol. Chem. 264, 12370]. Comparison of the apo, Mg(2+)-, and Ca(2+)-bound forms of cTnC3 and CBM-IIA demonstrates that the inability of CBM-IIA to trigger muscle contraction is not due to global structural changes in the mutant protein but is a consequence of the inability of CBM-IIA to bind Ca2+ at site II. The pattern of NOEs between aromatic residues in the C-terminal domain is nearly identical in cTnC3 and CBM-IIA. Similar interresidue NOEs were also observed between Phe residues assigned to the N-terminal domain in the Ca(2+)-saturated forms of both cTnC3 and CBM-IIA. However, chemical shift changes were observed for the N-terminal Phe residues in CBM-IIA. This suggests that binding of Ca2+ to site II alters the chemical environment of the residues in the N-terminal hydrophobic cluster without disrupting the spatial relationship between the Phe residues located in helices A and D.

Animals↗