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 t he hexameric repressor protein ArgR and the corepressor L-arginine. L- Arginine binds to ArgR in the C-terminal domain of the repressor. Bind ing to operator DNA occurs in the N-terminal domain. The molecular str uctures of both domains have recently been elucidated. The known stere ochemistry of the arginine binding pocket was used for the rational de sign of a mutant ArgR with altered ligand specificity. Our prediction was that a replacement of Asp128 by asparagine would preferentially le ad to the binding of L-citrulline, rather than L-arginine. The D128N m utant was constructed and was shown to fulfill our expectation by seve ral experimental approaches. By isothermal titration calorimetry it wa s found to bind L-citrulline much more strongly than L-arginine, in co ntrast to wildtype ArgR. Exchange between the mutant trimers of the he xamer was inhibited by L-citrulline, as it is by L-arginine in the wil d-type. The mutant protein was precipitated by L-citrulline but not by L-arginine, whereas the reverse is true for the wild-type protein. De monstration of a corepressor action was, however, precluded by the sup errepressor effect of the D128N mutation by itself. The mutant protein , in the absence of L-citrulline or L-arginine is as strong a represso r as the wild-type protein in the presence of L-arginine. We discuss t wo possible mechanisms, in terms of the known domain structures that c ould explain our observations. (C) 1998 Academic Press Limited.