Changing the Ligand Specificity of CooA, a Highly Specific Heme-based CO Sensor

Abstract

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.

CooA, a paradigm for gas sensing regulatory proteins

“…This then would create a driving force that helps reposition the C helices as detailed below. Similar experiments were performed on positions 117 and 120, with the result that the normal residue at each position, Gly117 and Leu120, is critical for CO-activation of CooA [37,58]. It appears likely that these residues have a role in C-helix repositioning, rather than in specific CO recognition, for reasons discussed in Section 5 below.…”

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

“…For example, O 2 forms a stable complex by hydrogen bonding to His-64 in myoglobin (41) and to Arg-220 in FixL (42). The tight distal heme pocket also precludes the activation of CooA by potential bulky ligands, such as imidazole (43). The formation of a tight trap for the functional ligand, which guarantees its fast geminate recombination with very high efficiency, is also seen in other heme-based sensor proteins, such as FixL (42).…”

Study of Highly Constitutively Active Mutants Suggests How cAMP Activates cAMP Receptor Protein

“…The CooA region homologous to CRP residues 125-130 on the C-helix was genetically randomized, and the resulting library of mutants was screened for those that activated CooA in the absence of the effector CO. Effector-independent CooA variants were readily found and uniformly possessed a consensus leucine zipper motif, consistent with the hypothesis (21). In another line of study, a novel CooA variant that is able to respond to the non-natural effector imidazole has been found, and the molecular basis for this is most easily explained by invoking a similar, but locally different, C-helix repositioning (22).…”