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

By H. Youn, R. Kerby, M. Conrad, G. Roberts. 2006

50 citations recorded by the source corpus.

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.

Subfields: Molecular Biology

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Citations in the corpus

Structural Basis of Dual Specificity of Sinorhizobium meliloti Clr, a cAMP and cGMP Receptor Protein (2023, background): “DNA binding domain in its active conformation (11, 54, 66, 67).”
Structural basis for cAMP-mediated allosteric control of the catabolite activator protein (2009, background): “In apo-CAP, Thr-127 and Ser-128 point away from the cAMP-binding pocket (Fig.”
Two-State Allosteric Modeling Suggests Protein Equilibrium as an Integral Component for Cyclic AMP (cAMP) Specificity in the cAMP Receptor Protein of Escherichia coli (2008, methodology): “codons were subsequently added at the 3 end as described previously (39).”
Structures of inactive CRP species reveal the atomic details of the allosteric transition that discriminates cyclic nucleotide second messengers. (2014, background): “Substitution of Thr127 and Ser128 by hydrophobic residues efficiently leads to the formation of a hydrophobic cluster at the hinge (Tzeng & Kalodimos, 2013; Youn et al., 2006; Chu et al., 2001).”
Neglected role of cAMP receptor protein monomer (2011, background): “Methyl and hydroxyl groups of T127 play essential role in stabilizing apo-CRP structure [16, 17].”
Directed Evolution of the Escherichia coli cAMP Receptor Protein at the cAMP Pocket* (2015, methodology): “The best example for the last case is the constitutively active T127L/S128I CRP mutant, which lacks hydrogen bonding ability at both positions, but possesses an extremely shifted protein equilibrium poise toward the active form (4).”