title: Repositioning about the dimer interface of the transcription regulator CooA: a major signal transduction pathway between the effector and DNA-binding domains.
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.
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, 106 E. B. Fred Hall, Madison, WI 53706, USA.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Kerby
authors.forename: Hwan
authors.initials: H
authors.lastname: Youn
authors.forename: Marc V
authors.initials: MV
authors.lastname: Thorsteinsson
authors.forename: Gary P
authors.initials: GP
authors.lastname: Roberts
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D013053:Spectrophotometry; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C108776:CooA protein, Rhodospirillum rubrum; D029968:Escherichia coli Proteins; D006420:Hemeproteins; D011994:Recombinant Proteins; D015534:Trans-Activators