The Role of the DNA-Binding Domains in CooA Activation

Abstract

Carbon monoxide oxidation activator protein (CooA) is a dimeric carbon monoxide (CO) binding transcription factor that in the presence of CO promotes the transcription of genes involved in CO oxidation in Rhodospirillum rubrum. The off state (inactive) of Fe(II) CooA has His and Pro as the two axial heme ligands. In contrast, in the on state, which is active in DNA binding, the Pro ligand bond has been replaced by CO. This occurs by the transient loss of the Pro ligand, thus generating a pentacoordinate heme that can bind CO. The active on state of CooA has two major structural differences from the off state, in addition to the displacement of the Pro ligand by CO. There is a repositioning of long C helices at the dimer interface and a concomitant reorientation of the hinge region between the DNA- and effector-binding domains within each monomer [Roberts et al. (2005) J. Inorg. Biochem. 99, 280-292]. To better understand the relationship of these conformational changes, we have removed the DNA-binding domains and compared CO binding to the truncated and full-length protein. The crystal structure of truncated Fe(II) CooA is the same as that of the effector-binding domain of full-length Fe(II) CooA, including the relative orientation of the homodimer and the ligation environment of the heme. Thus, removing the DNA-binding domains has little obvious effect on the structure of CooA in the inactive off state. However, CO binds about 2-fold more tightly and about 10 times more rapidly to truncated CooA. The rate of CO binding is known to be limited by the dissociation of the Pro heme ligand [Puranik et al. (2004) J. Biol. Chem. 279, 21096-21108]. Therefore, the absence of the DNA-binding domain makes it easier for the Pro ligand to dissociate from the heme iron, which also makes it easier for truncated CooA to adopt the on-state structure.

Effect of DNA Binding on Geminate CO Recombination Kinetics in CO-sensing Transcription Factor CooA

“…Fig. 2 shows the CO geminate recombination kinetics to both RrCooA and trun- cated RrCooA, lacking the DNA-binding domain (18), at room temperature. Note that the data are presented on a logarithmic scale.…”

“…This is in contrast with the crystal structure of the ferrous RrCooA that shows the C-helix of one monomer completely extended, whereas the other is kinked (6). Kuchinskas et al (18) explained this apparent discrepancy by suggesting that the off state may be characterized by an ensemble of DNA-binding domain orientations, whereas the DNA-bound on state has only one orientation. In this regard, the crystal structure of the inactive RrCooA can be considered as one of the several solution structures sampled by small angle x-ray scattering.…”

CO metabolism, sensing, and signaling

“…The best known CO-sensors is CooA, a homodimeric heme-containing protein, which regulates the CO oxidation system in the photosynthetic bacterium Rodospirillum rubrum [59][60][61][62], and the mammalian neuronal (N) period circadian protein (Per)-aryl hydrocarbon receptor nuclear translocator protein (Arnt) -single-minded protein (Sim) protein 2 (NPAS2), a member of the bHLH family of transcription factors expressed in the forebrain, which is a CO-dependent regulator of the circadian rhythm [59,60,63].…”

“…In the purple nonsulfur bacterium R. rubrum, in the presence of CO, CooA promotes the transcription of genes involved in CO oxidation [61,62]. Both chains of CooA contain a N-terminal sensory domain and a C-terminal DNA-binding domain, connected by the long C-helix [65].…”

“…The inactive ''off state'' of CooA has the His and Pro residues as the two axial ligands of the heme-Fe2 þ -atom. In contrast, in the ''on state,'' which is active in DNA binding, the Pro residue is replaced by CO [61,62]. In addition to CO binding, the active on state of CooA has two major structural differences from the off state: (i) the repositioning of the long C-helices at the dimer interface and (ii) the concomitant reorientation of the hinge region between the DNA-and effector-binding domains within each monomer [61].…”