Study of Highly Constitutively Active Mutants Suggests How cAMP Activates cAMP Receptor Protein

The cAMP receptor protein (CRP) of Escherichia coli undergoes a conformational change in response to cAMP binding that allows it to bind specific DNA sequences. Using an in vivo screening method following the simultaneous randomization of the codons at positions 127 and 128 (two C-helix residues of the protein interacting with cAMP), we have isolated a series of novel constitutively active CRP variants. Sequence analysis showed that this group of variants commonly possesses leucine or methionine at position 127 with a β-branched amino acid at position 128. One specific variant, T127L/S128I CRP, showed extremely high cAMP-independent DNA binding affinity comparable with that of cAMP-bound wild-type CRP. Further biochemical analysis of this variant and others revealed that Leu127 and Ile128 have different roles in stabilizing the active conformation of CRP in the absence of cAMP. Leu127 contributes to an improved leucine zipper at the dimer interface, leading to an altered intersubunit interaction in the C-helix region. In contrast, Ile128 stabilizes the proper position of the β4/β5 loop by functionally communicating with Leu61. By analogy, the results suggest two direct local effects of cAMP binding in the course of activating wild-type CRP: (i) C-helix repositioning through direct interaction with Thr127 and Ser128 and (ii) the concomitant reorientation of the β4/β5 loop. Finally, we also report that elevated expression of T127L/S128I CRP markedly perturbed E. coli growth even in the absence of cAMP, which suggests why comparably active variants have not been described previously.