A superrepressor mutant of the arginine repressor with a correctly predicted alteration of ligand binding specificity.
Arginine biosynthesis in Escherichia coli is negatively regulated by the hexameric repressor protein ArgR and the corepressor L-arginine. L-Arginine binds to ArgR in the C-terminal domain of the repressor. Binding to operator DNA occurs in the N-terminal domain. The molecular structures of both domains have recently been elucidated. The known stereochemistry of the arginine binding pocket was used for the rational design of a mutant ArgR with altered ligand specificity. Our prediction was that a replacement of Asp128 by asparagine would preferentially lead to the binding of L-citrulline, rather than L-arginine. The D128N mutant was constructed and was shown to fulfill our expectation by several experimental approaches. By isothermal titration calorimetry it was found to bind L-citrulline much more strongly than L-arginine, in contrast to wild-type ArgR. Exchange between the mutant trimers of the hexamer was inhibited by L-citrulline, as it is by L-arginine in the wild-type. The mutant protein was precipitated by L-citrulline but not by L-arginine, whereas the reverse is true for the wild-type protein. Demonstration of a corepressor action was, however, precluded by the superrepressor effect of the D128N mutation by itself. The mutant protein, in the absence of L-citrulline or L-arginine is as strong a repressor as the wild-type protein in the presence of L-arginine. We discuss two possible mechanisms, in terms of the known domain structures that could explain our observations.
Niersbach
H
H
Department of Microbiology, New York University Medical Center, 550 First Avenue, New York, NY, 10016, USA.
Lin
R
R
Van Duyne
G D
GD
Maas
W K
WK
D001120:Arginine / Q000096:biosynthesis / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D009154:Mutation; D011485:Protein Binding
D004247:DNA; D001120:Arginine