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 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.
Karaivanova
I M
IM
Laboratoire de Biotechnologie, UPRES Biocatalyse, Faculté des Sciences et des Techniques, Université de Nantes, Nantes, Cedex 3, 44322, France.
Weigel
P
P
Takahashi
M
M
Fort
C
C
Versavaud
A
A
Van Duyne
G
G
Charlier
D
D
Hallet
J N
JN
Glansdorff
N
N
Sakanyan
V
V
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000378:metabolism*; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D001411:Geobacillus stearothermophilus / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
C087018:ArgR protein, Bacteria; C490177:ArgR protein, E coli; D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D012097:Repressor Proteins