influenzae (Hi), E. coli (Ec), Vibrio cholerae (Vc), Pseudomonas putida (Pp), Rickettsia rickettsiae (Rr), Neisseria gonorrhoeae (Ng), Bdellovibrio bacteriovorus (Bba), Clostridium perfringens (Cp), Bacillus subtilis (Bs), Enterococcus faecalis (Ef), Streptococcus pneumoniae (Sp), Mycobacterium tuberculosis (Mt), Bacteroides capillosus (Bc), and B. burgdorferi (Bbu). Identical amino acids are boxed and shaded. Amino
acid residues of YbaBEc and YbaBHi that comprise αlpha-helices 1 and 3 of their determined protein structures are identified. After the genome sequence of H. influenzae strain KW20 rd SHP099 (also known as H. influenzae Rd) was determined in 1995 [2], the “”Structure 2 Function Project”" was established to crystallize recombinant proteins from H. influenzae genes of unknown function http://s2f.umbi.umd.edu/. Among these orphan gene
products was the H. influenzae DUF 149 group member annotated as open reading frame (ORF) HI0442, and tentatively named “”YbaB”" [3]. H. influenzae YbaB (YbaBHi) crystallized as a homodimer, with the central portion forming 3 antiparallel β-strands, long α-helices at the amino- and carboxy-termini (α-helices 1 and 3, respectively), and a short α-helix bridging the β-folded region and α-helix 3 (α-helix 2). The two subunits of the homodimer interface at the β-strand region, α-helix 2 and the initial residues of α-helix 3, while α-helix 1 and the terminal portion of α-helix 3 project away from the dimerization region. This distinctive structure that has been described as resembling a set of tweezers EPZ5676 [3]. Although the researchers who initially characterized YbaBHi speculated that it may be a DNA-binding protein, studies conducted at that time failed to detect binding to any of their BI 2536 analyzed DNA probes [3]. The Escherichia next coli chromosome carries an orthologous gene that has been referred to as “”ORF 12″” (Fig. 1) [4–6]. Recombinant E. coli YbaB (YbaBEc) has also been crystallized and information about its unpublished three-dimensional structure is available
on-line http://www.rcsb.org/pdb/explore.do?structureId=1PUG. The determined structures of YbaBEc and YbaBHi are nearly identical. A function for YbaBEc appears not to have been investigated prior to the current work. The spirochete Borrelia burgdorferi produces a protein named EbfC that shares 29% identical and 56% similar amino acids with YbaBHi (Fig. 1). Our laboratories recently discovered that EbfC binds a specific DNA sequence 5′ of the spirochete’s erp loci [7–10]. Those results suggested that orthologous proteins may also be DNA-binding proteins. We therefore re-examined the properties of YbaBHi, and found that it does bind to certain DNAs. YbaBEc was also demonstrated to be a DNA-binding protein. Results and discussion The abilities of YbaBEc and YbaBHi to bind DNA were first tested using a labeled DNA probe corresponding to sequences surrounding B. burgdorferi erpAB Operator 2 (Fig. 2).