RESUMO
The insecticidal Cry toxins produced by the bacterium Bacillus thuringiensis are comprised of three structural domains. Domain I, a seven-helix bundle, is thought to penetrate the insect epithelial cell plasma membrane through a hairpin composed of alpha-helices 4 and 5, followed by the oligomerization of four hairpin monomers. The alpha-helix 4 has been proposed to line the lumen of the pore, whereas some residues in alpha-helix 5 have been shown to be responsible for oligomerization. Mutation of the Cry1Ac1 alpha-helix 4 amino acid Asn135 to Gln resulted in the loss of toxicity to Manduca sexta, yet binding was still observed. In this study, the equivalent mutation was made in the Cry1Ab5 toxin, and the properties of both wild-type and mutant toxin counterparts were analyzed. Both mutants appeared to bind to M. sexta membrane vesicles, but they were not able to form pores. The ability of both N135Q mutants to oligomerize was also disrupted, providing the first evidence that a residue in alpha-helix 4 can contribute to toxin oligomerization.
Assuntos
Bacillus thuringiensis/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Mutação , Animais , Bacillus thuringiensis/química , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/toxicidade , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Endotoxinas/química , Endotoxinas/genética , Endotoxinas/metabolismo , Endotoxinas/toxicidade , Proteínas Hemolisinas , Manduca/efeitos dos fármacosRESUMO
Autoprocessing of the precursor form of human herpesvirus 6 (HHV-6) proteinase at two sites (termed M and R) is required to generate the mature enzyme. Kinetic constants were determined for the hydrolysis of a series of synthetic peptide substrates by mature HHV-6 proteinase, purified to homogeneity. Truncation or replacement of individual residues in peptides mimicking the R-site sequence, indicated that the minimum length for effective hydrolysis by the viral enzyme was P4-P3-P2-Ala*Ser-P2'-P3'-P4' and revealed the importance of the P1 Ala and P4 Tyr residues. Consequently, relevant (P1 or P4) mutations were introduced into the precursor form of the proteinase and the ability of these altered proteins to autoprocess was examined. Introduction of Val in place of the P1 Ala at the M-site essentially abrogated cleavage but mature HHV-6 proteinase was still generated by cleavage at the R-site, indicating that processing of the M-site is not a prerequisite for cleavage of the R-site in the precursor. At the R-site, mutation of the P1 Ala, or of the preceding P4 Tyr residue, prevented processing at the R-site in the precursor so that the mature form of HHV-6 proteinase was not generated. The accumulated data suggest a possible new approach to the design of inhibitors for therapeutic intervention in the life cycle of herpesviruses.
Assuntos
Endopeptidases/metabolismo , Precursores Enzimáticos/metabolismo , Herpesvirus Humano 6/enzimologia , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Precursores Enzimáticos/genética , Hidrólise , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Deleção de Sequência , Especificidade por SubstratoRESUMO
Amino acid residues thought to comprise the catalytic triad of HHV-6 proteinase were changed by site-directed mutagenesis in the precursor form of the proteinase. By monitoring the ability of each mutant proteinase precursor to undergo autoprocessing, Ser116, His46 and His135 were identified as catalytically crucial. An attempt was made to mimic the catalytic triad arrangement of archetypal serine proteinases by replacement of the second histidine, His135, by an Asp. Instead of increasing the autoprocessing ability of the His135Asp mutant HHV-6 proteinase precursor, this mutation had a detrimental effect since the precursor persisted predominantly in its unprocessed form.
Assuntos
Endopeptidases/metabolismo , Herpesvirus Humano 6/enzimologia , Sequência de Bases , Sítios de Ligação , Catálise , Primers do DNA , Endopeptidases/genética , Herpesvirus Humano 6/fisiologia , Hidrólise , Mutagênese Sítio-DirigidaRESUMO
After the U53 gene encoding the proteinase from human herpesvirus 6 (HHV-6) was sequenced, it was expressed in Escherichia coli, and the activity of the purified, recombinant HHV-6 proteinase was characterized quantitatively by using synthetic peptide substrates mimicking the release and maturation cleavage sites in the polyprotein precursors of HHV-6, human cytomegalovirus (CMV), murine CMV, and Epstein-Barr virus. Despite sharing 40% identity with other betaherpesvirus proteinases such as human CMV proteinase, the one-chain HHV-6 enzyme was distinguished from these two-chain proteinases by the absence of an internal autocatalytic cleavage site.
