Homology modeling of plant cytochrome P450s

Plant P450 monooxygenases represent the largest family of plant proteins and the largest collection of P450s available for comparative studies and biotechnological applications. They have been shown to catalyze a diverse array of difficult chemical reactions and have demonstrated potential to be used in pharmacological, agronomic and phytoremediative applications. Central to our use of these catalytically competent enzymes is the need to understand their interactions with substrates. Because most characterized plant P450s are membrane-bound proteins that are resistant to standard X-ray and NMR structure determinations, homology modeling represents a reliable and relatively rapid alternative method for analyzing structure-function relationships and predicting substrates for many P450s that are only now being characterized. These methods, which are being widely used in mammalian P450 structure-function studies, can allow plant biologists to define critical residues interacting with substrates and, in a directed fashion, alter the reactivities of individual monooxygenases. The homology modelings that have been done on a limited number of plant P450s and the site-directed mutations that validate them indicate that current modeling and substrate docking procedures are capable of providing structural explanations for sequence variants as well as for predicting functional characteristics of undefined P450s.     

Plasticity and steric strain in a parallel beta-helix: rational mutations in the P22 tailspike protein

By means of genetic screens, a great number of mutations that affect the folding and stability of the tailspike protein from Salmonella phage P22 have been identified. Temperature-sensitive folding (tsf) mutations decrease folding yields at high temperature, but hardly affect thermal stability of the native trimeric structure when assembled at low temperature. Global suppressor (su) mutations mitigate this phenotype. Virtually all of these mutations are located in the central domain of tailspike, a large parallel beta-helix. We modified tailspike by rational single amino acid replacements at three sites in order to investigate the influence of mutations of two types: (1) mutations expected to cause a tsf phenotype by increasing the side-chain volume of a core residue, and (2) mutations in a similar structural context as two of the four known su mutations, which have been suggested to stabilize folding intermediates and the native structure by the release of backbone strain, an effect well known for residues that are primarily evolved for function and not for stability or folding of the protein. Analysis of folding yields, refolding kinetics and thermal denaturation kinetics in vitro show that the tsf phenotype can indeed be produced rationally by increasing the volume of side chains in the beta-helix core. The high-resolution crystal structure of mutant T326F proves that structural rearrangements only take place in the remarkably plastic lumen of the beta-helix, leaving the arrangement of the hydrogen-bonded backbone and thus the surface of the protein unaffected. This supports the notion that changes in the stability of an intermediate, in which the beta-helix domain is largely formed, are the essential mechanism by which tsf mutations affect tailspike folding. A rational design of su mutants, on the other hand, appears to be more difficult. The exchange of two residues in the active site expected to lead to a drastic release of steric strain neither enhanced the folding properties nor the stability of tailspike. Apparently, side-chain interactions in these cases overcompensate for backbone strain, illustrating the extreme optimization of the tailspike protein for conformational stability. The result exemplifies the view arising from the statistical analysis of the distribution of backbone dihedral angles in known three-dimensional protein structures that the adoption of straight phi/psi angles other than the most favorable ones is often caused by side-chain interactions. Proteins 2000;39:89-101.     

Resistant cabbage cultivars change the susceptibility of Plutella xylostella to Bacillus thuringiensis

1 Laboratory studies demonstrated that the susceptibility of larvae of the lepidopteran crucifer pest Plutella xylostella to the insect pathogen Bacillus thuringiensis (Bt) was influenced by the host plant. 2 Larvae reared on the resistant cabbage cultivars Minicole F₁ and Red Drumhead were significantly more susceptible to Bt (the LC₅₀ fell to one half) than larvae fed leaves of susceptible cultivars. 3 However, a third resistant cultivar, Aquarius F₁, had no synergistic effect on Bt‐related mortality. 4 Actual uptake of Bt was monitored in the bioassays, as a preliminary experiment showed that the plant resistance reduced consumption of Bt‐treated leaf discs. However, differences in feeding rate did not explain the observed differences in mortality.     

Mit einem alkylierenden Agens (Zitostop) in vitro induzierbare Mutationen bei Malignomen und bei Syndromen,die zur Malignität disponieren

Zusammenfassung Die Zahl der mit einem alkylierenden Agens (Zitostop) in vitro induzierten Mutationen zeigt individuelle Schwankungen.Beim Down-Syndrom, dessen häufige Kombination mit malignen Erkrakungen bekannt ist, und bei 2 Mitgliedern einer Krebsfamilie wird eine statistisch signifikant erhöhte Zahl von induzierten Mutationen gegenüber Gesunden festgestellt.Bei der Ataxia teleangiektasia und bei akuter lymphoblastischer Leukämie im Remissionsstadium sind nur in einigen Fällen Chromosomenaberrationen in vitro gegenüber Normalen induzierbar. Nach unseren Beobachtungen stellt sich die Frage, ob die mit alkylierenden Agentien in vitro durchgeführten Untersuchungen zum Nachweis gesteigerten Mutationsneigung geeignet sind.

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Mutations induced in vitro by an alkylating agent (Zitostop) in malignant diseases and syndromes predisposing to malignancy

Summary According to our observations the number of chromosome mutations induced in vitro by an alkylating agent (Zitostop) shows individual variations. In Down's syndrome, which is accompanied by a higher incidence of malignancy, and in two members of a cancer family the number of mutations induced was significantly higher than in controls. In ataxia telangiectasia and in the complete remission phase of acute lymphoid leukaemia the number of mutations induced was higher only in some cases. The question of whether recording the number of chromosome mutations induced by an alkylating agent is a suitable procedure for the detection of an increased mutability or not is left open.

     

A natural variability in the proline-rich motif of Nef modulates HIV-1 replication in primary T cells

In the infected host, the Nef protein of HIV/SIV is required for high viral loads and thus disease progression. Recent evidence indicates that Nef enhances replication in the T cell compartment after the virus is transmitted from dendritic cells (DC). The underlying mechanism, however, is not clear. Here, we report that a natural variability in the proline-rich motif (R71T) profoundly modulated Nef-stimulated viral replication in primary T cells of immature dendritic cell/T cell cocultures. Whereas both Nef variants (R/T-Nef) downregulated CD4, only the isoform supporting viral replication (R-Nef) efficiently interacted with signaling molecules of the T cell receptor (TCR) environment and stimulated cellular activation. Structural analysis suggested that the R to T conversion induces conformational changes, altering the flexibility of the loop containing the PxxP motif and hence its ability to bind cellular partners. Our report suggests that functionally and conformationally distinct Nef isoforms modulate HIV replication on the interaction level with the TCR-signaling environment once the virus enters the T cell compartment.