Binding of CO at the Pro2 side is crucial for the activation of CO-sensing transcriptional activator CooA. (1)H NMR spectroscopic studies.
CooA is a heme-containing transcriptional activator that anaerobically binds to DNA at CO atmosphere. To obtain information on the conformational transition of CooA induced by CO binding to the heme, we assigned ring current-shifted (1)H NMR signals of CooA using two mutants whose axial ligands of the heme were replaced. In the absence of CO, the NMR spectral pattern of H77Y CooA, in which the axial histidine (His(77)) was replaced with tyrosine, was similar to that of wild-type CooA. In contrast, the spectra of CooADeltaN5, in which the NH(2) termini including the other axial ligand (Pro(2)) were deleted, were drastically modulated. We assigned three signals of wild-type CooA at -4.5, -3.6, and -2.8 ppm to delta(1)-, alpha-, and delta(2)-protons of Pro(2), respectively. The Pro(2) signals were undetectable in the upfield region of the spectrum of the CO-bound state, which confirms that CO displaces Pro(2). Interestingly, the Pro(2) signals were observed for CO-bound H77Y CooA, implying that CO binds to the trans position of Pro(2) in H77Y CooA. The abolished CO-dependent transcriptional activity of H77Y CooA is therefore the consequence of Pro(2) ligation. These observations are consistent with the view that the movement of the NH(2) terminus triggers the conformational transition to the DNA binding form.
Yamamoto
K
K
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan.
Ishikawa
H
H
Takahashi
S
S
Ishimori
K
K
Morishima
I
I
Nakajima
H
H
Aono
S
S
D001426:Bacterial Proteins; D002248:Carbon Monoxide / Q000378:metabolism*; D006420:Hemeproteins / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011392:Proline / Q000378:metabolism*; D011485:Protein Binding; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; C108776:CooA protein, Rhodospirillum rubrum; D006420:Hemeproteins; D015534:Trans-Activators; D002248:Carbon Monoxide; D011392:Proline