Repositioning about the dimer interface of the transcription regulator CooA: a major signal transduction pathway between the effector and DNA-binding domains.

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.