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M J Zvelebil

Publications and source records attributed to M J Zvelebil.

32 records · Page 2Linked to original sources
PubMed1994

Three-dimensional solution structure of the pleckstrin homology domain from dynamin.

BACKGROUND: The pleckstrin homology (PH) domain is a region of approximately 100 amino acids, defined by sequence similarity, that has been found in about 60 proteins, many of which are involved in signal transduction downstream of cell surface receptors; the function of PH domains is unknown. The only clue to the function of PH domains is the circumstantial evidence that they may link beta gamma subunits of G proteins to second messenger systems. Knowledge of the three-dimensional structures of PH domains should help to elucidate the roles they play in the proteins that contain them. RESULTS: Using homonuclear and heteronuclear magnetic resonance spectroscopy, we have determined the solution structure of the PH domain of the GTPase dynamin, one of a number of proteins that have PH domains and interact with GTP. The fold of the dynamin PH domain is composed of two antiparallel beta-sheets, which pack face-to-face at an angle of approximately 60 degrees. The first beta-sheet comprises four strands (residues 13-58) from the amino-terminal half of the protein sequence; the second beta-sheet contains three strands (residues 63-99). A single alpha-helix (residues 102-116) flanks one edge of the interface between the two sheets, parallel in orientation to the second sheet, in an alpha/beta roll motif similar to that of the B oligomer of verotoxin-1 from Escherichia coli. CONCLUSIONS: The structure of the dynamin PH domain is very similar to the recently reported structures of the pleckstrin and spectrin PH domains. This shows that, despite the low level of sequence similarity between different PH domains, they do have a characteristic polypeptide fold. On the basis of our structure, the suggestion that PH domains engage in coiled-coil interactions with G protein beta gamma subunits seems unlikely and should be re-evaluated.

Amino Acid Sequence↗
PubMed1994

Mechanism of inhibition of trypanothione reductase and glutathione reductase by trivalent organic arsenicals.

The dithiol trypanothione, novel to trypanosomatids and analogous to glutathione in mammalian systems, has been shown to interact with anti-trypanocidal trivalent arsenical drugs forming a stable adduct, MelT. This adduct is a competitive inhibitor of the flavoprotein trypanothione reductase, responsible for maintaining intracellular trypanothione in the reduced form. Since trypanothione reductase and the analogous glutathione reductase both contain catalytically active sulphydryl groups we have examined the ability of several arsenicals to differentially inhibit these enzymes. Melarsen oxide [p-(4,6-diamino-s-triazin-2-yl)aminophenylarsenoxide] potently inhibits both enzymes in two stages, the first being essentially complete within 1 min, the second being time dependent, exhibiting saturable pseudo-first-order kinetics with kinact of 14.3 x 10(-4) s-1 and 1.06 x 10(-4) s-1 and Ki of 17.2 microM and 9.6 microM for trypanothione reductase and glutathione reductase, respectively. Inhibition requires prior reduction of the enzyme by NADPH and can be reversed by excess dithiols or prevented by MelT in the case of trypanothione reductase. In both cases a time-dependent loss of the characteristic charge-transfer absorbance band at 530 nm is observed upon addition of arsenical to pre-reduced enzyme, which with excess NADPH leads to a spectrum resembling the EH4 form and is accompanied by an increased ability to reduce molecular oxygen. A model for inhibition is proposed where, first, free arsenical and previously reduced enzyme immediately establish an equilibrium with an inactive monothioarsane enzyme-inhibitor complex involving the interchange cysteine distal to the FAD; second, a subsequent rearrangement about the sulphur-arsenic bond leads to the binding of the arsenical to the charge-transfer cysteine, proximal to the FAD, forming a more stable dithioarsane complex. Molecular modelling suggests that the differences in kinetic behaviour of the two enzymes can be attributed to structural features of their respective disulphide-binding sites. Incubation of reduced trypanothione reductase with excess dihydrotrypanothione and melarsen oxide prevents direct inhibition of the enzyme, suggesting that dihydrotrypanothione acts as a protectant in vivo, preventing the direct modification of trypanothione reductase by sequestering the arsenical as MelT.

Animals↗
PubMed1993

Molecular modelling and epitope prediction of gp29 from lymphatic filariae.

The sequence of the major soluble protein component of the cuticle of filarial nematodes is homologous to that of bovine glutathione peroxidase, for which an X-ray structure is available. Due to the high degree of sequence identity (42%), it has been possible to build an apparently reliable three-dimensional model of the gp29 cuticular protein from Brugia spp. that will aid studies of the molecule both as a target immunogen and secreted enzyme. The modelled core of the gp29 structure is conserved compared to the bovine enzyme, consisting of a beta-sheet surrounded by alpha-helices. Experimental data showed that Brugia spp. gp29 has four subunits, and a tetrameric form of gp29 has also been modelled. The two N-linked glycosylation sites per subunit were predicted to lie on the surface of the tetramer. Most of the variation in amino acid sequence compared to that of mammalian enzymes, occurs in the surface loops, several of which are larger and more exposed in gp29. Deglycosylated gp29 was demonstrated to be immunogenic in human infection, and six likely B-cell epitopes have been predicted on the basis of a high protrusion index and sequence variability.

Amino Acid Sequence↗
PubMed1993

A detailed molecular model for human aromatase.

Using a variety of techniques, including sequence alignment, secondary structure prediction, molecular mechanics and molecular dynamics, we have constructed a model for the three-dimensional structure of P-450arom (human aromatase) based on that of P-450cam, the only cytochrome P-450 enzyme for which the crystal structure is known. The predicted structure is found to be in good agreement with current experimental data; both direct, from site-directed mutagenesis studies, and indirect, from the consideration of the structures and activities of known substrates and inhibitors.

Amino Acid Sequence↗
PubMed1991

A predicted three-dimensional structure of human cytochrome P450: implications for substrate specificity.

A three-dimensional structure for human cytochrome P450IA1 was predicted based on the crystal coordinates of cytochrome P450cam from Pseudomonas putida. As there was only 15% residue identity between the two enzymes, additional information was used to establish an accurate sequence alignment that is a prerequisite for model building. Twelve representative eukaryotic sequences were aligned and a net prediction of secondary structure was matched against the known alpha-helices and beta-sheets of P450cam. The cam secondary structure provided a fixed main-chain framework onto which loops of appropriate length from the human P450IA1 structure were added. The model-built structure of the human cytochrome conformed to the requirements for the segregation of polar and nonpolar residues between the core and the surface. The first 44 residues of human cytochrome P450 could not be built into the model and sequence analysis suggested that residues 1-26 formed a single membrane-spanning segment. Examination of the sequences of cytochrome P450s from distinct gene families suggested specific residues that could account for the differences in substrate specificity. A major substrate for P450IA1, 3-methyl-cholanthrene, was fitted into the proposed active site and this planar aromatic molecule could be accommodated into the available cavity. Residues that are likely to interact with the haem were identified. The sequence similarity between 59 eukaryotic enzymes was represented as a dendrogram that in general clustered according to gene family. Until a crystallographic structure is available, this model-building study identifies potential residues in cytochrome P450s important in the function of these enzymes and these residues are candidates for site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1991

A pyruvate-stimulated adenylate cyclase has a sequence related to the fes/fps oncogenes and to eukaryotic cyclases.

The pyruvate-stimulated adenylate cyclase from Brevibacterium liquefaciens produces up to 450 microM cyclic AMP in the culture medium when the bacterium is grown on glucose and alanine. In this paper we report the cloning, expression and sequencing of the gene for this enzyme. Residues were identified, within the C-terminal domain, which are conserved in adenylate and guanylate cyclase sequences from eukaryotes and in the adenylate cyclase of the prokaryote Rhizobium meliloti. We have also identified a sequence of 30 residues near the N-terminus of the protein which is homologous to part of the regulatory domain of the cellular homologues of the oncogenes fes and fps; this sequence is also present in the avian Fujinami sarcoma virus fps gene.

Adenylyl Cyclases↗
PubMed1990

A molecular model for the enzyme cytochrome P450(17 alpha), a major target for the chemotherapy of prostatic cancer.

The enzyme cytochrome P450(17 alpha) catalyses two key steps in the biosynthesis of the androgens from pregnanes: the 17 alpha hydroxylation step and the subsequent 17-20 lyase reaction. Using a variety of techniques, including sequence alignment, secondary structure prediction, molecular mechanics and molecular dynamics, we have constructed a model for the three-dimensional structure of P450(17 alpha) based on that of P450cam, the only cytochrome P450 enzyme for which the crystal structure is known. The model suggests the possibility of two modes of binding of steroid substrates at the active site, perhaps reflecting the dual functionality of the enzyme.

Amino Acid Sequence↗
PubMed1990

Prediction of protein structure from sequence.

Methods to predict the three-dimensional structure of a protein from its sequence are reviewed. The approaches to derive information about the local conformation from the local sequence include hydrophobicity plots, secondary structure prediction and the identification of short, functional sequence motifs. The most reliable method of tertiary structure prediction is model building from the experimentally determined coordinates of a protein with an homologous sequence. This approach is illustrated by a prediction of the three-dimensional structure of human cytochrome P450-IA1. If no known homologous structure is available, then the only approach is to suggest models for the tertiary fold of proteins by packing together predicted secondary structures. A three-dimensional model for the dimerisation of the transmembrane alpha-helices of neu, a tyrosine kinase growth factor receptor, is described. In general, structure prediction can suggest approaches for regulating protein activity that may lead to new pharmaceutical therapies for cancer.

Amino Acid Sequence↗
PubMed1988

Predictions of linear T-cell and B-cell epitopes in proteins encoded by HIV-1, HIV-2 and SIVMAC and the conservation of these sites between strains.

An important consideration in the design of vaccines to prevent HIV-1 infection effective against different strains is the amino acid sequence conservation of antigenic determinants. Even one amino acid change can destroy the antigenicity of a site for the antibody or T-cell receptor. The comparisons of predicted T- and B-cell epitopes between human HIV-1, HIV-2 and monkey SIVMAC AIDS viruses are presented. The three major gene products (env, gag and pol) were examined. A number of epitopes were identical between strains of HIV-1. Our analysis highlights the problem of designing an effective HIV-1 and HIV-2 vaccine and also the problem of testing human vaccines in monkey models.

Amino Acid Sequence↗
PubMed1988

Analysis and prediction of the location of catalytic residues in enzymes.

The catalytic residues of an enzyme are defined as the amino acids directly involved in chemical catalysis. They mainly act as a general acid--base, electrophilic or nucleophilic catalyst or they polarize and stabilize the transition state. An analysis of the structural features of 36 catalytic residues in 17 enzymes of known structure and with defined mechanism is reported. Residues that bind metal ions (Zn2+ and Cu2+) are considered separately. The features examined are: residue type, location in secondary structure, separation between the residues, accessibility to solvent, intra-protein electrostatic interactions, mobility as evaluated from crystallographic temperature factors, polarity of the environment and the sequence conservation between homologous enzymes of residues that were sequentially or spatially close to the catalytic residue. In general the environment of catalytic residues is similar to that of polar side chains that have low accessibility to solvent. Two algorithms have been developed to identify probable catalytic residues. Scanning an alignment of homologous enzyme sequences for peaks of sequence conservation identifies 13 out of the 16 catalytic residues with 50 residues overpredicted. When the conservation of the spatially close residues is used instead, a different set of 13 residues are identified with 47 residues overpredicted. A combination of the two algorithms identifies 11 residues with 36 residues overpredicted.

Algorithms↗
PubMed1987

Prediction of antigenic determinants and secondary structures of the major AIDS virus proteins.

Criteria for the design of peptide vaccines to prevent AIDS are presented. The best vaccine candidates contain both B and T lymphocyte-defined epitopes in regions conserved in sequence between viral isolates. We propose that attention should focus on proteins specified by the gag and, possibly, pol genes in addition to the env gene envelope glycoproteins being actively studied. The predictions of B- and T-epitopes are refined by consideration of secondary structure prediction and inter-isolate sequence variability to suggest peptides from env, gag and pol that would be the best vaccine candidates.

Amino Acid Sequence↗
PubMed1987

Prediction of protein secondary structure and active sites using the alignment of homologous sequences.

The prediction of protein secondary structure (alpha-helices, beta-sheets and coil) is improved by 9% to 66% using the information available from a family of homologous sequences. The approach is based both on averaging the Garnier et al. (1978) secondary structure propensities for aligned residues and on the observation that insertions and high sequence variability tend to occur in loop regions between secondary structures. Accordingly, an algorithm first aligns a family of sequences and a value for the extent of sequence conservation at each position is obtained. This value modifies a Garnier et al. prediction on the averaged sequence to yield the improved prediction. In addition, from the sequence conservation and the predicted secondary structure, many active site regions of enzymes can be located (26 out of 43) with limited over-prediction (8 extra). The entire algorithm is fully automatic and is applicable to all structural classes of globular proteins.

Algorithms↗