MUTAGENESIS OF THE CYCLIC-AMP RECEPTOR PROTEIN OF ESCHERICHIA-COLI - TARGETING POSITION-83, POSITION-127 AND POSITION-128 OF THE CYCLIC-NUCLEOTIDE BINDING POCKET

The cyclic 3', 5' adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coil was mutagenized to su bstitute cysteine or glycine for serine 83; cysteine, glycine, isoleuc ine, or serine for threonine 127; and threonine or alanine for serine 128. Cells that expressed the binding pocket residue-substituted forms of CRP were characterized by measurements of beta-galactosidase activ ity. Purified wild-type and mutant CRP preparations were characterized by measurement of cAMP binding activity and by their capacity to supp ort lacP activation in vitro. CRP structure was assessed by measuremen t of sensitivity to protease and DTNB-mediated subunit crosslinking. T he results of this study show that cAMP interactions with serine 83, t hreonine 127 and serine 128 contribute to CRP activation and have litt le effect on cAMP binding. Amino acid substitutions that introduce hyd rophobic amino acid side chain constituents at either position 127 or 128 decrease CRP discrimination of cAMP and cGMP. Finally, cAMP-induce d CRP structural change(s) that occur in or near the CRP hinge region result from cAMP interaction with threonine 127; substitution of threo nine 127 by cysteine, glycine, isoleucine, or serine produced forms of CRP that contained, independently of cAMP binding, structural changes similar to those of the wildtype CRP:cAMP complex.