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J. Biol. Chem., Vol. 281, Issue 2, 1119-1127, January 13, 2006

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

From the Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706

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 -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 4/5 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 4/5 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.

Received for publication, August 25, 2005 , and in revised form, September 29, 2005.

^* The work was supported by the College of Agricultural and Life Sciences of the University of Wisconsin at Madison and by National Institutes of Health Grant GM53228 (to G. P. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Bacteriology, University of Wisconsin, 420 Henry Mall, Madison, WI 53706. Tel.: 608-262-3567; Fax: 608-262-9865; E-mail: groberts{at}bact.wisc.edu.

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