YbiB from Escherichia coli, the Defining Member of the Novel TrpD2 Family of Prokaryotic DNA-binding Proteins

Abstract

We present the crystal structure and biochemical characterization of Escherichia coli YbiB, a member of the hitherto uncharacterized TrpD2 protein family. Our results demonstrate that the functional diversity of proteins with a common fold can be far greater than predictable by computational annotation. The TrpD2 proteins show high structural homology to anthranilate phosphoribosyltransferase (TrpD) and nucleoside phosphorylase class II enzymes but bind with high affinity (KD = 10-100 nM) to nucleic acids without detectable sequence specificity. The difference in affinity between single- and double-stranded DNA is minor. Results suggest that multiple YbiB molecules bind to one longer DNA molecule in a cooperative manner. The YbiB protein is a homodimer that, therefore, has two electropositive DNA binding grooves. But due to negative cooperativity within the dimer, only one groove binds DNA in in vitro experiments. A monomerized variant remains able to bind DNA with similar affinity, but the negative cooperative effect is eliminated. The ybiB gene forms an operon with the DNA helicase gene dinG and is under LexA control, being induced by DNA-damaging agents. Thus, speculatively, the TrpD2 proteins may be part of the LexA-controlled SOS response in bacteria.

Keywords: DNA damage response; DNA-binding protein; cooperativity; crystallography; dimerization; functional annotation.

FIGURE 1.

Phylogenetic analysis of the TrpD, TrpD2, and NP-II protein families. Representatives were selected from the TrpD, TrpD2 (YbiB), and NP-II families that cover the full range of sequence variation within each family. The tree was analyzed by bootstrapping, and values are given at each edge.

FIGURE 2.

Analytical size exclusion chromatography of wild-type YbiB and the mutant variant YbiB_L40E,I51E. 100 μl of a 20 μm protein solution (subunit concentration) were applied to a calibrated Superdex S75 column and eluted in 50 mm potassium phosphate, pH 7.5, 300 mm NaCl at a flow rate of 0.5 ml/min, which was followed by the absorption at 280 nm. The on-column concentration of both variants is ∼1 μm (corresponding to 22 milliabsorbance units). The elution volumes of the peaks correspond to molecular masses of 61.9 kDa (WT; calculated dimeric molecular mass of 71.8 kDa) and 33.7 kDa (YbiB_L40E,I51E; calculated monomeric molecular mass of 35.9 kDa).

FIGURE 3.

Structural features of YbiB. A, ribbon diagram of the dimeric TrpD2 protein from E. coli, YbiB. The protomers are colored in blue and orange, and the helices that constitute the contact interface are labeled. B, surface electrostatic view of the YbiB dimer, showing negatively charged (red) and positively charged (blue) potentials. The two views are rotated against each other by ∼75°. The magnified section shows the dimer contact interface in a semitransparent view. Arg and Lys residues that contribute to the charged groove from the second (top) protomer are shown in a stick representation. C, comparison of the active site region of TrpD (S. solfataricus TrpD, Protein Data Bank code 1ZYK), NP-II (Geobacillus stearothermophilus PYNP, Protein Data Bank code 1BRW), and TrpD2 (E. coli YbiB). Co-crystallized ligands are colored in red: AA-I, AA-II, and PRPP (TrpD) and uracil and P_i (NP-II). The residues discussed under “Results” are colored as follows: conserved Arg (gray); glycine-rich loop TGG or YNG (green); His from KH, KHGN, or HG (magenta); highly conserved His in TrpD2 (cyan). The α1-α2 loop in TrpD2 is labeled, and Arg and Lys residues that contribute to the charged groove from the second (top) protomer are shown in a stick representation. The point of view is similar to that for the magnified section in B.

FIGURE 4.

A, superposition of YbiB (protomers in orange and blue) with TrpD from S. solfataricus (purple; Protein Data Bank code 1O17). Superposition was done separately for the N- and C-terminal domain of each protomer to compensate hinge movements between the domains. The root mean square deviation is 1.6 Å for 60 C_α atoms in the N-terminal domain and 2.6 Å for 148 C_α atoms in the C-terminal domain. B, surface electrostatic view of YbiB. The tryptophan residue at the rim of the charged groove that was used for fluorescence studies is colored in green. C, space-filling model of YbiB in two views, rotated against each other by ∼75°. Residues are colored in a red (100%) to white (0%) gradient according to their degree of conservation, derived from the TrpD2 alignment shown in Fig. 5. The color gradient was generated with protskin (43) and is based on sequence identity. Note that the angles of view are identical to those for Fig. 3; the angle of view of the left panel is identical to A, and the angle of view of the right panel is identical to B.

FIGURE 5.

Multiple sequence alignment of the TrpD and TrpD2 families, shown as sequence logos. The E. coli YbiB sequence is shown as a reference. All Arg and Lys residues in E. coli YbiB that contribute to the charged groove are colored in blue. The conserved GTGGD motif in TrpD, representing the PRPP binding loop, and the residues involved in the binding of anthranilic acid (A) are marked in orange at the top. The sequence logo algorithm fades out columns that are represented in a few sequences only, resulting in seemingly empty gap columns. In consequence, gap columns indicate variable protein regions in this representation.

FIGURE 6.

Electrophoretic mobility shift assays of YbiB, using dsDNA and ssDNA probes of different length (A–F). Different concentrations of protein were preincubated in 50 mm Tris/HCl, 2 mm potassium phosphate, pH 7.5, with constant amounts of the radioactively labeled probes of random sequence and analyzed on native 6 or 10% polyacrylamide gels. The protein subunit concentrations used (μm) are displayed above each lane. The signal of the free probe in each lane was quantified by densitometry and plotted against protein concentration (graphs below the EMSA autoradiographs). The Hill equation was fitted to the data. K_D values and Hill coefficients (n) were derived from the fit and are given for each graph.

FIGURE 7.

Analysis of the YbiB-DNA interaction by fluorescence titration experiments. E. coli YbiB (A–E) or aaTrpD2 (F), 2 μm monomer concentration each, were titrated with different DNA or RNA oligonucleotides. The length (in bases) and type of oligonucleotide are shown in the legend (dT, oligo(dT) ssDNA; random, random ssDNA; dsDNA, random dsDNA; U, oligo(U) RNA). The normalized fluorescence is plotted against the concentration of the oligonucleotide. Most experiments were repeated three times (except for D, 20b random, dT), and error bars (S.D.) are given. In the case of aaTrpD2 (F), the absolute fluorescence signal and signal change were lower (4% change versus 7–21% in the case of YbiB, depending on probe length). The Trp located in the binding groove is missing in aaTrpD2, but one of the two distal Trp residues gives some signal. This results in less homogeneous curves.

FIGURE 8.

Influence of increasing salt concentrations on the binding of YbiB to ssDNA. YbiB was titrated with a 12-base-long dT oligonucleotide in 50 mm Tris, pH 7.5, plus different concentrations of potassium phosphate (A) or 50 mm Tris, 2 mm potassium phosphate, pH 7.5, plus different concentrations of NaCl (B). K_D values were calculated from the titration curves and plotted against the salt concentration. All data points are the mean of three experiments. Error bars, S.D.

FIGURE 9.

Fluorescence titration of WT YbiB and the monomerized variant YbiB_L40E,I51E with dT oligonucleotides of different length. The amount of protein was 2 μm (monomer concentration) for each titration. Each curve represents the mean of three experiments. The data for WT YbiB are identical to those for Fig. 7A; for details see the legend to Fig. 7. The calculated affinities and stoichiometries are summarized in Tables 5 and 6. Error bars, S.D.

FIGURE 10.

Operon organization of ybiB. A, genomic organization of ybiB and surrounding genes. The dinG promoter (gray box) is controlled by the LexA repressor. Primer pairs 1–4 used for RT-PCR are depicted as arrows. B, RT-PCR analysis to test for co-transcription of ybiB and its neighbors. Primer pairs 1 and 3 are positive controls and test for intact mRNAs containing dinG or ybiB. Primer pair 2 tests for co-transcription of dinG and ybiB, and primer pair 4 tests for co-transcription of ybiB and ybiC.

FIGURE 11.

Influence of mutagenic agents on ybiB expression. Increasing amounts of mitomycin C (MMC; given in μg/ml above each lane) were added during cultivation. Equal amounts of total cellular protein were separated by SDS-PAGE, blotted and YbiB (36 kDa) detected by an anti-His₆ antibody (Roche Applied Science). The GAPDH protein (36 kDa) was used as a loading control.