Mutagenesis of cyclic AMP receptor protein: targeting positions 72 and 82 of the cyclic nucleotide binding pocket

The cyclic adenosine 3',5' monophosphate (cAMP): cAMP receptor protein (CRP) complex functions to promote transcription activation of several operons in Escherichia coli. The complex binds to specific DNA sites located upstream of, for example, the lactose operon promoter (lacP). This activates lacP and allows high level expression of lactose operon structural gene sequences. Five specific amino acid residues are predicted to play a role in the interaction of cAMP with CRP. The purpose of this work was to test the role of two amino acid residues, glutamate at position 72 and arginine at position 82, in facilitating both cAMP binding and cAMP-mediated activation of CRP.

Mutants at positions 072 and 082 in the cyclic nucleotide binding pocket of the CRP were constructed by site directed mutagenesis. Glutamate at position 072 was substituted by either leucine, glutamine, or aspartate. Arginine at the position 082 was substituted by either lysine, histidine, leucine, isoleucine, or glutamine. A parallel set of substitutions was constructed in a CRP*, or cAMP-independent, form of the protein to determine the effects of the amino acid substitutions on CRP conformation. Each mutant form of CRP was characterized in vivo with respect to sugar utilization, and through quantitative pgalactosidase activity and in vitro by measurement of cAMP binding activity under near physiological salt concentrations.

The results of this study confirm a role for both glutamate at position 72 and arginine at position 82 in cAMP binding and activation of CRP; neither aspartate at position 72 nor lysine at position 82 could effectively substitute for glutamate or arginine in an otherwise wild-type CRP. The results of this study also indicate that cGMP, a competitive inhibitor of cAMP binding to CRP, binds the cyclic nucleotide binding pocket differendy than cAMP.