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

Recently the crystal structure of the DNA-unbound form of the full-length h exameric Bacillus stearothermophilus arginine repressor (ArgR) has been res olved, providing a possible explanation for the mechanism of arginine-media ted repressor-operator DNA recognition. In this study we tested some of the se functional predictions by performing site-directed mutagenesis of distin ct amino acid residues located in two regions, the N-terminal DNA-binding d omain and the C-terminal oligomerization domain of ArgR. A total of 15 muta nts were probed for their capacity to repress the expression of the reporte r argC-lacZ gene fusion in Escherichia coli cells. Substitutions of highly conserved amino acid residues in the alpha 2 and alpha 3 helices, located i n the winged helix-turn-helix DNA-binding motif, reduced repression. Loss o f 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. st earothermophilus ArgR molecule behaves as a super-repressor, since recombin ant E. coli host cells bearing B. stearothermophilus argR on a multicopy ve ctor did not grow in selective minimal medium devoid of arginine and grew, albeit weakly, when L-arginine was supplied. All mutants affected in the DN A-binding domain lost this super-repressor behaviour. Replacements of conse rved leucine residues at positions 87 and/or 94 in the C-terminal domain by other hydrophobic amino acid residues proved neutral or caused either dere pression or stronger super-repression. Substitution of Leu87 by phenylalani ne was found to increase the DNA-binding affinity and the protein solubilit y in the context of a double Leu87Phe/Leu94Val mutant. Structural modificat ions occasioned by the various amino acid substitutions were confirmed by c ircular dichroism analysis and structure modelling. (C) 1999 Academic Press .