title: Characterization of variants altered at the N-terminal proline, a novel heme-axial ligand in CooA, the CO-sensing transcriptional activator.
abstract: CooA, the carbon monoxide-sensing transcription factor from Rhodospirillum rubrum, binds CO through a heme moiety resulting in conformational changes that promote DNA binding. The crystal structure shows that the N-terminal Pro(2) of one subunit (Met(1) is removed post-translationally) provides one ligand to the heme of the other subunit in the CooA homodimer. To determine the importance of this novel ligand and the contiguous residues to CooA function, we have altered the N terminus through two approaches: site-directed mutagenesis and regional randomization, and characterized the resulting CooA variants. While Pro(2) appears to be optimal for CooA function, it is not essential and a variety of studied variants at this position have substantial CO-sensing function. Surprisingly, even alterations that add a residue (where Pro(2) is replaced by Met(1)-Tyr(2), for example) accumulate heme-containing CooA with functional properties that are similar to those of wild-type CooA. Other nearby residues, such as Phe(5) and Asn(6) appear to be important for either the structural integrity or the function of CooA. These results are contrasted with those previously reported for alteration of the His(77) ligand on the opposite side of the heme.
authors.affiliation: Department of Bacteriology and the Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
authors.forename: M V
authors.initials: MV
authors.lastname: Thorsteinsson
authors.forename: R L
authors.initials: RL
authors.lastname: Kerby
authors.forename: M
authors.initials: M
authors.lastname: Conrad
authors.forename: H
authors.initials: H
authors.lastname: Youn
authors.forename: C R
authors.initials: CR
authors.lastname: Staples
authors.forename: W N
authors.initials: WN
authors.lastname: Lanzilotta
authors.forename: T J
authors.initials: TJ
authors.lastname: Poulos
authors.forename: J
authors.initials: J
authors.lastname: Serate
authors.forename: G P
authors.initials: GP
authors.lastname: Roberts
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002474:Cell-Free System; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D036781:Fimbriae Proteins; D005454:Fluorescence Polarization; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011392:Proline / Q000737:chemistry*; D011487:Protein Conformation; D013053:Spectrophotometry; D015533:Transcriptional Activation; D014466:Ultraviolet Rays; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C088904:CooA protein, E coli; D029968:Escherichia coli Proteins; D008024:Ligands; D036781:Fimbriae Proteins; D011392:Proline; D001616:beta-Galactosidase