PMID:12507482
| Citation |
Kerby, RL, Youn, H, Thorsteinsson, MV and Roberts, GP (2003) Repositioning about the dimer interface of the transcription regulator CooA: a major signal transduction pathway between the effector and DNA-binding domains. J. Mol. Biol. 325:809-23 |
|---|---|
| Abstract |
Activation of the homodimeric transcriptional regulator CooA depends on the coupling of CO binding at an effector domain heme with the allosteric repositioning of the DNA-binding domain F-helix that promotes specific DNA interaction. By analogy to the homologous cAMP receptor protein (CRP), it has been proposed that effector binding elicits subunit reorientation about their coiled-coil C-helix interface, and that this effector domain reorientation stabilizes the active position of the DNA-binding domains. Here, we describe experiments in which effector-independent "CooA*" variants were selected following randomization of a six-residue portion of the C-helix dimerization domain. Subsequent activity analyses, both in vivo and in vitro, were consistent with a model wherein improved C-helix "leucine zipper" interactions modestly shifted the regulator population equilibrium towards the active conformation, although full activation remained CO-dependent. However, in addition to the improved leucine zipper, maximal CooA* activity required additional C-helix changes which in a WT background decreased normal CO-dependent DNA-binding 100-fold. This seemingly paradoxical combination suggested that maximal CooA* activity depended both on the improved coiled-coil interactions and the decoupling of the signal pathway within the effector domain. Both types of C-helix changes indicate that its repositioning is crucial for the allosteric shift in the inactive/active equilibrium of the DNA-binding domain. |
| Links | |
| Keywords |
Amino Acid Sequence; Bacterial Proteins; Binding Sites; Dimerization; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Hemeproteins/chemistry; Hemeproteins/genetics; Hemeproteins/metabolism; Models, Molecular; Molecular Sequence Data; Mutagenesis; Protein Conformation; Protein Structure, Quaternary; Protein Structure, Tertiary; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Sequence Homology, Amino Acid; Signal Transduction; Spectrophotometry; Trans-Activators/chemistry; Trans-Activators/genetics; Trans-Activators/metabolism |
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