Mutational analysis of the thermostable arginine repressor from Bacillus stearothermophilus: dissecting residues involved in DNA binding properties.

Autor: Karaivanova IM; Laboratoire de Biotechnologie, UPRES Biocatalyse, Faculté des Sciences et des Techniques, Université de Nantes, Nantes, Cedex 3, 44322, France., Weigel P, Takahashi M, Fort C, Versavaud A, Van Duyne G, Charlier D, Hallet JN, Glansdorff N, Sakanyan V
Jazyk: angličtina
Zdroj: Journal of molecular biology [J Mol Biol] 1999 Aug 27; Vol. 291 (4), pp. 843-55.
DOI: 10.1006/jmbi.1999.3016
Abstrakt: Recently the crystal structure of the DNA-unbound form of the full-length hexameric Bacillus stearothermophilus arginine repressor (ArgR) has been resolved, providing a possible explanation for the mechanism of arginine-mediated repressor-operator DNA recognition. In this study we tested some of these functional predictions by performing site-directed mutagenesis of distinct amino acid residues located in two regions, the N-terminal DNA-binding domain and the C-terminal oligomerization domain of ArgR. A total of 15 mutants were probed for their capacity to repress the expression of the reporter argC - lacZ gene fusion in Escherichia coli cells. Substitutions of highly conserved amino acid residues in the alpha2 and alpha3 helices, located in the winged helix-turn-helix DNA-binding motif, reduced repression. Loss of DNA-binding capacity was confirmed in vitro for the Ser42Pro mutant which showed the most pronounced effect in vivo. In E. coli, the wild-type B. stearothermophilus ArgR molecule behaves as a super-repressor, since recombinant E. coli host cells bearing B. stearothermophilusargR on a multicopy vector did not grow in selective minimal medium devoid of arginine and grew, albeit weakly, when l -arginine was supplied. All mutants affected in the DNA-binding domain lost this super-repressor behaviour. Replacements of conserved leucine residues at positions 87 and/or 94 in the C-terminal domain by other hydrophobic amino acid residues proved neutral or caused either derepression or stronger super-repression. Substitution of Leu87 by phenylalanine was found to increase the DNA-binding affinity and the protein solubility in the context of a double Leu87Phe/Leu94Val mutant. Structural modifications occasioned by the various amino acid substitutions were confirmed by circular dichroism analysis and structure modelling.
(Copyright 1999 Academic Press.)
Databáze: MEDLINE
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