Functionally critical elements of CooA-related CO sensors

Abstract

CooA is a heme-containing transcriptional activator that enables Rhodospirillum rubrum to sense and grow on CO as a sole energy source. We have identified a number of CooA homologs through database searches, expressed these heterologously in Escherichia coli, and monitored their ability to respond to CO in vivo. Further in vitro analysis of two CooA homologs from Azotobacter vinelandii and Carboxydothermus hydrogenoformans corroborated the in vivo data by revealing the ability of CO to bind to these hemoproteins and stimulate their binding at specific DNA sequences. These data, as well as the patterns of conserved residues in the homologs, are compared to what is already known about functionally important residues in the CooA protein of R. rubrum. The results identify critical regions of CooA and indicate features that distinguish CooAs from the general family of cyclic AMP receptor proteins.

FIG. 1.

Inactive Fe(II) CooA structure adapted from that of the strain with PDB identification no. 1FT9. The protein consists of two monomers, shaded differently in this figure, which dimerize along the central C-helices of adjacent effector-binding domains. The solved structure is asymmetric, in which one monomer contains fused C- and D-helices (20). Nonetheless, both F-helices that interact with DNA in a sequence-specific manner are buried from the surface in the structure. The 4/5 loop is noted and so are the Pro2 and His77 heme Fe(II) ligands. In these representations, the DNA-binding domains form the upper part of each structure and the positions of the helices that specifically interact with DNA are designated as the F-helix.

FIG. 2.

Sequence alignment of CooA homologs. Eight CooA homologs and CRP from E. coli were aligned by using the T-Coffee multiple sequence alignment tool (version 1.37) as implemented at http://www.cmbi.kun.nl/bioinf/tools/T_COFFEE/ (25). Putative CooA homologs were identified by a TBLASTN search of 207 completed and unfinished microbial genomes available at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi) queried with the R. rubrum CooA protein. Designations of the aligned sequencesand their accession parameters are as follows: Rr, R. rubrum (gi no. 1498752); Dh(2), D. hafniense (gi no. 23114031); Ch2350, C. hydrogenoformans (gnl no. TIGR_129958, contig 2350:c_hydrogenoformans); Av, A. vinelandii (gi no. 23105899); Dd, D. desulfuricans G20 (gi no. 23473780); Dv, D. vulgaris (gnl no. TIGR_881, 1531); Dh, D. hafniense (gi no. 23111778); Ch2340, C. hydrogenoformans (gnl no. TIGR_129958, contig 2340:c_hydrogenoformans); Ec CRP, E. coli (gi no. 117484). Shaded portions indicate 70% conservation, with key structural elements indicated above sections of the alignment. These include residues that form the distal and proximal heme ligand environments (overlined with a dotted line) as well as conserved structural elements including the His heme ligand (His77 in R. rubrum CooA), the β-4 sheet, helices αC, αE, and αF, the hinge sequence between the effector and DNA-binding domains, and the AR1 and AR3 regions that interact with RNA polymerase (overlined with a thick line). Boldface underlined residues at or near the N terminus represent the cloned initiation of the proteins; for the CooA from R. rubrum, the terminal Met is removed posttranscriptionally.

FIG. 3.

Cells containing most CooA homologs accumulate heme-containing proteins. CooA proteins were partially purified from anaerobically grown cells by the hydroxylapatite preparation described in Materials and Methods. The spectrum of each partially purified sample was corrected by subtracting the spectrum of a similarly treated control sample of the same protein concentration prepared from a strain containing only the pEXT20 vector. OD, optical density.

FIG. 4.

UV-visible spectra of the selected CooA homologs. (A) R. rubrum CooA; (B) C. hydrogenoformans 2340 CooA; (C) C80S C. hydrogenoformans 2340 CooA; (D) A. vinelandii CooA. CooA proteins were partially purified from anaerobically grown cells by the hydroxylapatite preparation described in Materials and Methods. Analyzed samples were diluted to 1 mg of protein/ml. For the spectra of C. hydrogenoformans 2340 CooA proteins, 20 μM potassium ferricyanide [K₃Fe(CN)₆] was added to the as isolated forms of the proteins.

FIG. 5.

In vitro DNA-binding activities of selected CooA homologs purified from anaerobically grown cells. The analyzed samples duplicated those prepared for spectral analyses (Fig. 4). The activities of the CooA homologs were measured at the same concentrations of either heme or protein. −, absent.