Biomedical subjects

I Fita

Publications and source records attributed to I Fita.

At least 37 records · Page 2Linked to original sources

PubMed1999

The structures of picornaviral proteinases.

Picornaviruses are a family of positive-strand RNA viruses the members of which include poliovirus, hepatitis A virus, rhinovirus, foot-and-mouth disease virus and encephalomyocarditis virus. The genetic information contained in the single-stranded, positive sense RNA genome is expressed as a single protein of around 2000 amino acids. This primary product of protein synthesis, designated the polyprotein, is subsequently cleaved into the mature viral proteins by proteinases present within it. The properties of the three defined proteolytic activities present in the picornaviruses are reviewed and the three-dimensional structures of the hepatitis A 3C proteinase and the leader proteinase of foot-and-mouth disease virus as well as a model of the structure of the HRV2 2A proteinase are compared with those of chymotrypsin, papain and streptomyces griseus A proteinase, respectively.

3C Viral Proteases↗

PubMed1999

Crystallization and preliminary structural results of catalase from human erythrocytes.

Catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, E.C. 1. 11.1.6) is present in most aerobic prokaryotic and eukaryotic cells. Despite a large number of studies on catalases, the only mammalian catalase structure available is that from beef liver, in which about 50% of the haem groups are degraded to bile pigments. Three different crystal forms of human erythrocyte catalase were obtained by the hanging-drop vapour-diffusion technique using PEG as precipitant. Monoclinic crystals, with space group P21 and unit-cell parameters a = 102.9, b = 140.0, c = 173.6 A and beta = 103.2 degrees, require NADP(H) in the crystallization solution. Two types of hexagonal packing, with unit-cell parameters of either a = b = 86. 9, c = 255.5 A or a = b = 90.0, c = 521.2 A, were obtained under identical crystallization conditions in the absence of NADP(H). Only one diffraction data set could be collected: this was obtained from the hexagonal crystals with the smaller c axis using synchrotron radiation, with resolution to 2.65 A. A molecular-replacement solution, determined using a modified beef-liver catalase model as a search structure, corresponds to space group P6422 and contains a single subunit in the asymmetric unit, with an estimated solvent volume of about 50%. The packing determined suggests how minor rearrangements might allow the transition between both hexagonal crystal forms and provides an explanation for the anisotropic character of the corresponding diffractions.

Catalase↗

PubMed1999

N-Acetyl-L-glutamate kinase from Escherichia coli: cloning of the gene, purification and crystallization of the recombinant enzyme and preliminary X-ray analysis of the free and ligand-bound forms.

The gene for Escherichia coli N-acetyl-L-glutamate kinase (NAGK) was cloned in a plasmid and expressed in E. coli, allowing enzyme purification in three steps. NAGK exhibits high specific activity (1.1 micromol s-1 mg-1), lacks Met1 and forms dimers (shown by cross-linking). Crystals of unliganded NAGK diffract to 2 A and belong to space group P6122 or its enantiomorph P6522 (unit-cell parameters a = b = 78.6, c = 278.0 A) with two monomers in the asymmetric unit. Crystals of NAGK with acetylglutamate and the ATP analogue AMPPNP diffract to 1.8 A and belong to space group C2221 (unit-cell parameters a = 60.0, b = 71.9, c = 107.4 A), with one monomer in the asymmetric unit. NAGK crystallization will allow the determination of proposed structural similarities to carbamate kinase.

Base Sequence↗

PubMed1999

Crystallization and preliminary X-ray analysis of human rhinovirus serotype 2 (HRV2).

Human rhinoviruses, the major cause of mild recurrent infections of the upper respiratory tract, are small icosahedral particles. Over 100 different serotypes have been identified. The majority (91 serotypes) use intercellular adhesion molecule 1 as the cell-attachment site; ten serotypes (the minor group) bind to members of the low-density lipoprotein receptor. Three different crystal forms of the minor-group human rhinovirus serotype 2 (HRV2) were obtained by the hanging-drop vapour-diffusion technique using ammonium sulfate and sodium/potassium phosphate as precipitants. Monoclinic crystals, space group P2(1), diffracted at least to 2.8 A resolution, and two complete virus particles were located in the crystal asymmetric unit. A second type of crystals had a compact cubic like morphology and diffracted beyond 2.5 A resolution. These crystals belong to a primitive orthorhombic space group, with unit-cell parameters a = 309.3, b = 353.5, c = 759.6 A, and contain one virus particle in the asymmetric unit. A third type of crystals, with a prismatic shape and belonging to space group I222, was also obtained under similar crystallization conditions. These latter crystals, with unit-cell parameters a = 308.7, b = 352.2, c = 380.5 A, diffracted to high resolution (beyond 1.8 A) and contained 15 protomers per asymmetric unit; this requires that three perpendicular crystal twofold axes coincide with three of the viral particle's dyad axes.

Crystallization↗

PubMed1999

Role of the lateral channel in catalase HPII of Escherichia coli.

The heme-containing catalase HPII of Escherichia coli consists of a homotetramer in which each subunit contains a core region with the highly conserved catalase tertiary structure, to which are appended N- and C-terminal extensions making it the largest known catalase. HPII does not bind NADPH, a cofactor often found in catalases. In HPII, residues 585-590 of the C-terminal extension protrude into the pocket corresponding to the NADPH binding site in the bovine liver catalase. Despite this difference, residues that define the NADPH pocket in the bovine enzyme appear to be well preserved in HPII. Only two residues that interact ionically with NADPH in the bovine enzyme (Asp212 and His304) differ in HPII (Glu270 and Glu362), but their mutation to the bovine sequence did not promote nucleotide binding. The active-site heme groups are deeply buried inside the molecular structure requiring the movement of substrate and products through long channels. One potential channel is about 30 A in length, approaches the heme active site laterally, and is structurally related to the branched channel associated with the NADPH binding pocket in catalases that bind the dinucleotide. In HPII, the upper branch of this channel is interrupted by the presence of Arg260 ionically bound to Glu270. When Arg260 is replaced by alanine, there is a threefold increase in the catalytic activity of the enzyme. Inhibitors of HPII, including azide, cyanide, various sulfhydryl reagents, and alkylhydroxylamine derivatives, are effective at lower concentration on the Ala260 mutant enzyme compared to the wild-type enzyme. The crystal structure of the Ala260 mutant variant of HPII, determined at 2.3 A resolution, revealed a number of local structural changes resulting in the opening of a second branch in the lateral channel, which appears to be used by inhibitors for access to the active site, either as an inlet channel for substrate or an exhaust channel for reaction products.

Alanine↗

PubMed1999

Carbamate kinase: New structural machinery for making carbamoyl phosphate, the common precursor of pyrimidines and arginine.

The enzymes carbamoyl phosphate synthetase (CPS) and carbamate kinase (CK) make carbamoyl phosphate in the same way: by ATP-phosphorylation of carbamate. The carbamate used by CK is made chemically, whereas CPS itself synthesizes its own carbamate in a process involving the phosphorylation of bicarbonate. Bicarbonate and carbamate are analogs and the phosphorylations are carried out by homologous 40 kDa regions of the 120 kDa CPS polypeptide. CK can also phosphorylate bicarbonate and is a homodimer of a 33 kDa subunit that was believed to resemble the 40 kDa regions of CPS. Such belief is disproven now by the CK structure reported here. The structure does not conform to the biotin carboxylase fold found in the 40 kDa regions of CPS, and presents a new type of fold possibly shared by homologous acylphosphate-making enzymes. A molecular 16-stranded open beta-sheet surrounded by alpha-helices is the hallmark of the CK dimer. Each subunit also contains two smaller sheets and a large crevice found at the location expected for the active center. Intersubunit interactions are very large and involve a central hydrophobic patch and more hydrophilic peripheral contacts. The crevice holds a sulfate that may occupy the site of an ATP phosphate, and is lined by conserved residues. Site-directed mutations tested at two of these residues inactivate the enzyme. These findings support active site location in the crevice. The orientation of the crevices in the dimer precludes their physical cooperation in the catalytic process. Such cooperation is not needed in the CK reaction but is a requirement of the mechanism of CPSs.

Arginine↗

PubMed1998

Structure of the foot-and-mouth disease virus leader protease: a papain-like fold adapted for self-processing and eIF4G recognition.

The leader protease of foot-and-mouth disease virus, as well as cleaving itself from the nascent viral polyprotein, disables host cell protein synthesis by specific proteolysis of a cellular protein: the eukaryotic initiation factor 4G (eIF4G). The crystal structure of the leader protease presented here comprises a globular catalytic domain reminiscent of that of cysteine proteases of the papain superfamily, and a flexible C-terminal extension found intruding into the substrate-binding site of an adjacent molecule. Nevertheless, the relative disposition of this extension and the globular domain to each other supports intramolecular self-processing. The different sequences of the two substrates cleaved during viral replication, the viral polyprotein (at LysLeuLys/GlyAlaGly) and eIF4G (at AsnLeuGly/ArgThrThr), appear to be recognized by distinct features in a narrow, negatively charged groove traversing the active centre. The structure illustrates how the prototype papain fold has been adapted to the requirements of an RNA virus. Thus, the protein scaffold has been reduced to a minimum core domain, with the active site being modified to increase specificity. Furthermore, surface features have been developed which enable C-terminal self-processing from the viral polyprotein.

Amino Acid Sequence↗

PubMed1998

Solution conformation of an immunogenic peptide from HRV2: comparison with the conformation found in a complex with a Fab fragment of an anti-HRV2 neutralizing antibody.

The conformation of a [15]-peptide (H-VKAETRLNPDLQPTE-NH2) from VP2 of rhinovirus HRV2 complexed with a Fab fragment was previously shown by X-ray crystallographic studies to be similar to the one found in the corresponding region of HRV1A. Antibodies raised against this peptide bind to and neutralize HRV2. In order to identify structural features preserved in solution that may explain the ability of this short peptide to mimic the structure of the protein surface, the peptide has been studied by NMR in aqueous solution as well as under denaturing conditions. The peptide is shown to be a random coil in solution. However, the sequence forming a 3(10)helix in the complex is biased into a helical conformation according to NOE intensity data as well as from urea and pH titrations. This sequence adopts the same conformation in an unrelated protein. NOE data suggest that a beta-turn found in the complex may be sampled in solution. Also, Glu4, interacting with Arg6 in the crystal, has a reduced pKa value in solution. It is concluded that the local structure present in the random coil state of VP2(156-170) contains enough information to direct the production of antibodies that bind to and neutralize HRV2.

Amino Acid Sequence↗

PubMed1998

Truncation and heme pocket mutations reduce production of functional catalase HPII in Escherichia coli.

The subunit of catalase HPII from Escherichia coli is 753 residues in length and contains a core of approximately 500 residues, with high structural similarity to all other heme catalases. To this core are added extensions of approximately 80 and 180 residues at the N- and C-termini, respectively. The tetrameric structure is made up of a pair of interwoven dimers in which 90 N-terminal residues of each subunit are inserted through a loop formed by the hinge region linking the beta-barrel and alpha-helical domains of the adjacent subunit. A high concentration of proline residues is found in the vicinity of the overlap regions. To study the influence of the extended regions on folding and subunit association of HPII, a diversity of modifications have been introduced. Removal of the complete C-terminal domain or the N-terminal extension, either separately or together, effectively creating a small subunit catalase, resulted in no enzyme accumulation. Systematic truncations showed that only nine C-terminal residues (Ile745 to Ala753) could be removed without significantly affecting the accumulation of active enzyme. Removal or even conservative replacements of the side chain of Arg744 significantly reduced the accumulation of active enzyme despite this residue interacting only with the C-terminal domain. Removal of as few as 18 residues from the N-terminus also reduced accumulation of active enzyme. Changes to other residues in the protein, including residues in the heme binding pocket, also reduced the accumulation of active protein without substantially affecting the enzyme specific activity. Implications of these data for the interdependence of subunit folding and subunit-subunit interactions are discussed.

Catalase↗

PubMed1998

A structural model of picornavirus leader proteinases based on papain and bleomycin hydrolase.

The leader (L) proteinases of aphthoviruses (foot-and-mouth disease viruses) and equine rhinovirus serotypes 1 and 2 cleave themselves from the growing polyprotein. This cleavage occurs intramolecularly between the C terminus of the L proteinases and the N terminus of the subsequent protein VP4. The foot-and-mouth disease virus enzyme has been shown, in addition, to cleave at least one cellular protein, the eukaryotic initiation factor 4G. Mechanistically, inhibitor studies and sequence analysis have been used to classify the L proteinases as papain-like cysteine proteinases. However, sequence identity within the L proteinases themselves is low (between 18% and 32%) and only 14% between the L proteinases and papain. Secondary structure predictions, sequence alignments that take into account the positions of the essential catalytic residues, and structural considerations have been used in this study to investigate more closely the relationships between the L proteinases and papain. In spite of the low sequence identities, the analyses strongly suggest that the L proteinases of foot-and-mouth disease virus and of equine rhinovirus 1 have a similar overall fold to that of papain. Regions in the L proteinases corresponding to all five alpha-helices and seven beta-sheets of papain could be identified. Further comparisons with the proteinase bleomycin hydrolase, which also displays a papain topology in spite of important differences in size and amino acid sequence, support these conclusions and suggest how a C-terminal extension, present in all three L proteinases, and predicted to be an alpha-helix, might enable C-terminal self-processing to occur.

Amino Acid Sequence↗

PubMed1998

A similar pattern of interaction for different antibodies with a major antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation.

The three-dimensional structures of the Fab fragment of a neutralizing antibody raised against a foot-and-mouth disease virus (FMDV) of serotype C1, alone and complexed to an antigenic peptide representing the major antigenic site A (G-H loop of VP1), have been determined. As previously seen in a complex of the same antigen with another antibody which recognizes a different epitope within antigenic site A, the receptor recognition motif Arg-Gly-Asp and some residues from an adjacent helix participate directly in the interaction with the complementarity-determining regions of the antibody. Remarkably, the structures of the two antibodies become more similar upon binding the peptide, and both undergo considerable induced fit to accommodate the peptide with a similar array of interactions. Furthermore, the pattern of reactivities of five additional antibodies with versions of the antigenic peptide bearing amino acid replacements suggests a similar pattern of interaction of antibodies raised against widely different antigens of serotype C. The results reinforce the occurrence of a defined antigenic structure at this mobile, exposed antigenic site and imply that intratypic antigenic variation of FMDV of serotype C is due to subtle structural differences that affect antibody recognition while preserving a functional structure for the receptor binding site.

Amino Acid Sequence↗

PubMed1997

Structure of the complex of an Fab fragment of a neutralizing antibody with foot-and-mouth disease virus: positioning of a highly mobile antigenic loop.

Data from cryo-electron microscopy and X-ray crystallography have been combined to study the interactions of foot-and-mouth disease virus serotype C (FMDV-C) with a strongly neutralizing monoclonal antibody (mAb) SD6. The mAb SD6 binds to the long flexible GH-loop of viral protein 1 (VP1) which also binds to an integrin receptor. The structure of the virus-Fab complex was determined to 30 A resolution using cryo-electron microscopy and image analysis. The known structure of FMDV-C, and of the SD6 Fab co-crystallized with a synthetic peptide corresponding to the GH-loop of VP1, were fitted to the cryo-electron microscope density map. The SD6 Fab is seen to project almost radially from the viral surface in an orientation which is only compatible with monovalent binding of the mAb. Even taking into account the mAb hinge and elbow flexibility, it is not possible to model bivalent binding without severely distorting the Fabs. The bound GH-loop is essentially in what has previously been termed the 'up' position in the best fit Fab orientation. The SD6 Fab interacts almost exclusively with the GH-loop of VP1, making very few other contacts with the viral capsid. The position and orientation of the SD6 Fab bound to FMDV-C is in accord with previous immunogenic data.

Antibodies, Monoclonal↗

PubMed1997

Crystallization and preliminary structural analysis of catalase A from Saccharomyces cerevisiae.

Yeast peroxisomal catalase A, obtained at high yields by over expression of the C-terminally modified gene from a 2 mu-plasmid, has been crystallized in a form suitable for high resolution X-ray diffraction studies. Brownish crystals with bipyrimidal morphology and reaching ca. 0.8 mm in size were produced by the hanging drop method using ammonium sulphate as precipitant. These crystals diffract better than 2.0 A resolution and belong to the hexagonal space group P6(1)22 with unit cell parameters a = b = 184.3 A and c = 305.5 A. An X-ray data set with 76% completeness at 3.2 A resolution was collected in a rotating anode generator using mirrors to improve the collimation of the beam. An initial solution was obtained by molecular replacement only when using a beef liver catalase tetramer model in which fragments with no sequence homology had been omitted, about 150 residues per subunit. In the structure found a single molecule of catalase A (a tetramer with accurate 222 molecular symmetry) is located in the asymmetric unit of the crystal with an estimated solvent content of about 61%. The preliminary analysis of the structure confirms the absence of a carboxy terminal domain as the one found in the catalase from Penicillium vitalae, the only other fungal catalase structure available. The NADPH binding site appears to be involved in crystal contacts, suggesting that heterogeneity in the occupancy of the nucleotide can be a major difficulty during crystallization.

Catalase↗

PubMed1997

Identification of a novel bond between a histidine and the essential tyrosine in catalase HPII of Escherichia coli.

A bond between the N delta of the imidazole ring of His 392 and the C beta of the essential Tyr 415 has been found in the refined crystal structure at 1.9 A resolution of catalase HPII of Escherichia coli. This novel type of covalent linkage is clearly defined in the electron density map of HPII and is confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis of tryptic digest mixtures. The geometry of the bond is compatible with both the sp3 hybridization of the C beta atom and the planarity of the imidazole ring. Two mutated variants of HPII active site residues, H128N and N201H, do not contain the His 392-Tyr 415 bond, and their crystal structures show that the imidazole ring of His 392 was rotated, in both cases, by 80 degrees relative to its position in HPII. These mutant forms of HPII are catalytically inactive and do not convert heme b to heme d, suggesting a relationship between the self-catalyzed heme conversion reaction and the formation of the His-Tyr linkage. A model coupling the two processes and involving the reaction of one molecule of H2O2 on the proximal side of the heme with compound 1 is proposed.

Amino Acid Sequence↗

PubMed1997

The structure of alfalfa mosaic virus capsid protein assembled as a T=1 icosahedral particle at 4.0-A resolution.

K. Fukuyama, S. S. Abdel-Meguid, J. E. Johnson, and M. G. Rossmann (J. Mol. Biol. 167:873-984, 1983) reported the structure of alfalfa mosaic virus assembled from the capsid protein as a T=1 icosahedral empty particle at 4.5-A resolution. The information contained in the structure included the particle size, protein shell thickness, presence of wide holes at the icosahedral fivefold axes, and a proposal that the capsid protein adopts a beta-barrel structure. In the present work, the X-ray diffraction data of Fukuyama et al. as well as the data subsequently collected by I. Fita, Y. Hata, and M. G. Rossmann (unpublished) were reprocessed to 4.0-A resolution, and the structure was solved by molecular replacement. The current structure allowed the tracing of the polypeptide chain of the capsid protein confirming the beta-sandwich fold and provides information on intersubunit interactions in the particle. However, it was not possible to definitively assign the amino acid sequence to the side chain density at 4-A resolution. The particle structure was also determined by cryoelectron microscopy and image reconstruction methods and found to be in excellent agreement with the X-ray model.

Alfalfa mosaic virus↗

PubMed1997

Efficient neutralization of foot-and-mouth disease virus by monovalent antibody binding.

Neutralization of an aphthovirus by monovalent binding of an antibody is reported. Foot-and-mouth disease virus (FMDV) clone C-S8c1 was neutralized by monoclonal antibody (MAb) SD6, which was directed to a continuous epitope within a major antigenic site of the G-H loop of capsid protein VP1. On a molar basis, the Fab fragment was at most fivefold less active in neutralization than the intact antibody, and both blocked virus attachment to cells. Neither the antibody nor the Fab fragment caused aggregation of virions, as evidenced by sucrose gradient sedimentation studies of the antibody-virus complex formed at antibody to virion ratios of 1:50 to 1:10,000. The results of neutralization of infectivity and of ultracentrifugation are fully consistent with structural data based on X-ray crystallographic and cryoelectron microscopy studies, which showed monovalent interaction of the antibody with a critical receptor binding motif Arg-Gly-Asp. The conclusions of these neutralization studies are that (i) bivalent binding of antibody is not a requisite for strong neutralization of aphthoviruses and (ii) aggregation of viral particles, which has been proposed to be the dominant neutralization mechanism of antibodies that bind monovalently to virions, is not necessary for the neutralization of FMDV C-S8c1 by MAb SD6.

Animals↗

PubMed1996

Structure of the heme d of Penicillium vitale and Escherichia coli catalases.

A heme d prosthetic group with the configuration of a cis-hydroxychlorin gamma-spirolactone has been found in the crystal structures of Penicillium vitale catalase and Escherichia coli catalase hydroperoxidase II (HPII). The absolute stereochemistry of the two heme d chiral carbon atoms has been shown to be identical. For both catalases the heme d is rotated 180 degrees about the axis defined by the alpha-gamma-meso carbon atoms, with respect to the orientation found for heme b in beef liver catalase. Only six residues in the heme pocket, preserved in P. vitale and HPII, differ from those found in the bovine catalase. In the crystal structure of the inactive N201H variant of HPII catalase the prosthetic group remains as heme b, although its orientation is the same as in the wild type enzyme. These structural results confirm the observation that heme d is formed from protoheme in the interior of the catalase molecule through a self-catalyzed reaction.

Catalase↗

PubMed1996

Induced pocket to accommodate the cell attachment Arg-Gly-Asp motif in a neutralizing antibody against foot-and-mouth-disease virus.

The three-dimensional structure of the Fab fragment of a neutralizing monoclonal antibody (SD6) elicited against foot-and-mouth disease virus (FMDV) has been determined at 2.5 A resolution and refined to a crystallography agreement R-factor of 0.186. The structure has been compared with that of the same Fab molecule complexes with a 15 amino acid peptide (A15) representing a major antigenic site of FMDV, and determined at 2.8 A resolution. The Fab quaternary structure, defined both by the elbow angle between modules and by the relative disposition of the light and heavy domains inside the modules, remains essentially unchanged. However, the comparison shows important conformational variations in the paratope, especially in the hypervariable loops of the heavy chain. The CDR-H3 loop has a peculiar amino acid sequence (RREDGGDEGF) with a high content of charged residues. Some of these Fab residues were fully reoriented upon complex formation. The reorientation resulted not only in an alteration of shape but also in an important redistribution of charges, providing multiple points of interaction with the A15 antigen and in particular with the cell attachment Arg-Gly-Asp motif in the peptide. Thus the recognition of A15 by SD6 represents an extreme example of the induced fit mechanism in antibody interactions. The electron density maps provide evidence that in the uncomplexed Fab structure some CDR residues show, with lower occupancy, the conformations found in the complex, suggesting that the rearrangements observed can have only minor energetic requirements.

Amino Acid Sequence↗