Changing the ligand specificity of CooA, a highly specific heme-based CO sensor.
The CO-specific heme-based sensor CooA regulates the ability of Rhodospirillum rubrum to grow on CO as an energy source. Only CO triggers the conformational change of CooA essential for the protein to function as a transcriptional activator. A structurally informed mutagenesis, followed by an in vivo screening method, allowed the isolation of a series of novel CooA variants that show very substantial response to imidazole. Compared with wild-type CooA, the ligand selectivity between imidazole and CO had been changed in some variants by roughly three orders of magnitude. Remarkably, different CooA variants also showed the ability to discriminate among imidazole derivatives, strongly implying a mechanism of precise interactions between the affected residues and the various ligands. Although wild-type CooA and imidazole-responsive CooA variants appear to recognize their respective ligands by fundamentally different mechanisms, several lines of evidence suggest that they respond by a similar C-helix repositioning that results in the rearrangement of the DNA-binding domains responsible for specific DNA contact. These results have implications for the molecular basis of both the imidazole responsiveness in the variants and the stringent CO specificity of wild-type CooA.
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Hwan
H
Youn
Robert L
RL
Kerby
Gary P
GP
Roberts
D002248:Carbon Monoxide / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D036781:Fimbriae Proteins / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D007093:Imidazoles / Q000378:metabolism; D008024:Ligands
C088904:CooA protein, E coli; D029968:Escherichia coli Proteins; D007093:Imidazoles; D008024:Ligands; D036781:Fimbriae Proteins; D006418:Heme; C029899:imidazole; D002248:Carbon Monoxide; D004247:DNA