Regulation of single-stranded DNA binding by the C termini of Escherichia coli single-stranded DNA-binding (SSB) protein

Abstract

The homotetrameric Escherichia coli single-stranded DNA-binding (SSB) protein plays a central role in DNA replication, repair, and recombination. In addition to its essential activity of binding to transiently formed single-stranded (ss) DNA, SSB also binds an array of partner proteins and recruits them to their sites of action using its four intrinsically disordered C-terminal tails. Here we show that the binding of ssDNA to SSB is inhibited by the SSB C-terminal tails, specifically by the last 8 highly acidic amino acids that comprise the binding site for its multiple partner proteins. We examined the energetics of ssDNA binding to short oligodeoxynucleotides and find that at moderate salt concentration, removal of the acidic C-terminal ends increases the intrinsic affinity for ssDNA and enhances the negative cooperativity between ssDNA binding sites, indicating that the C termini exert an inhibitory effect on ssDNA binding. This inhibitory effect decreases as the salt concentration increases. Binding of ssDNA to approximately half of the SSB subunits relieves the inhibitory effect for all of the subunits. The inhibition by the C termini is due primarily to a less favorable entropy change upon ssDNA binding. These observations explain why ssDNA binding to SSB enhances the affinity of SSB for its partner proteins and suggest that the C termini of SSB may interact, at least transiently, with its ssDNA binding sites. This inhibition and its relief by ssDNA binding suggest a mechanism that enhances the ability of SSB to selectively recruit its partner proteins to sites on DNA.

FIGURE 1.

ssDNA wrapping around the SSB tetramer in its (SSB)₆₅ mode is unaffected by its C-terminal tails. A, model depicting 65 nucleotides of ssDNA (red ribbon) wrapped around the four SSB subunits, consisting of four OB-folds (shown in cyan, green, yellow, and gray), based on the x-ray crystallographic structure of the SSB_c tetramer bound to two molecules of (dC)₃₅ (20) with the addition of the unstructured C-terminal tails (shown in gray) that are not observed in the crystal structure. The 9 amino acids at the end of each C terminus, responsible for the interaction of SSB with other metabolic proteins, is shown in single letter amino acid codes. B, results of fluorescence titrations of 0.14 μm wtSSB (●) and SSB_C (○) (lacking 42 amino acids from its C termini) with poly(dT) (buffer H, pH 8.1, 0.2 m NaCl, 25 °C). The linear increase in relative Trp fluorescence quenching indicates stoichiometric binding of the protein to poly(dT). The occluded site sizes were determined by extrapolation of the linear part of the titration curve to the point of intersection with the plateau as described (12) and yields n ∼ 63 nucleotides/tetramer for SSB_C and n ∼ 65 for wtSSB.

FIGURE 2.

Salt dependence of ssDNA binding to SSBΔC8 and wtSSB. A–D, results of Trp fluorescence titrations of SSB (●) and SSBΔC8 (○) (0.1–0.2 μm) with (dA)₃₅ at 20 mm NaCl (A), 0.1 m NaCl (B), and 0.2 m NaCl (C) and with (dT)₃₅ in 0.6 m NaBr (D) (buffer T, pH 8.1, 25 °C). The smooth curves in panels B, C, and D represent the best fits of the data to either a two-site sequential binding model or a square model (Equations 1–4 under “Experimental Procedures”) with the parameters shown in Table 1.

FIGURE 3.

ITC studies of the effect of temperature on the inhibition of ssDNA binding by the SSB C termini. All studies were performed in buffer T, pH 8.1, 100 mm NaCl. A, 15 °C, ITC titrations of wtSSB (■) (0.9 μm) with (dA)₃₅ (21 μm). The smooth curve represents the best fit of the experimental isotherm to Equations 5 and 6 (under “Experimental Procedures”) with the following parameters: n = 0.86 ± 0.03, K_obs = (2.2 ± 0.5) × 10⁷ m⁻¹, and ΔH_obs = 10.9 ± 0.5 kcal/mol. B, 15 °C, ITC titrations of SSBΔC8 (0.3 μm (●) and 1.8 μm (▴) with (dA)₃₅ (7.5 and 35 μm, respectively). The smooth curves represent the best fit of the experimental isotherms to a two-site sequential binding model (Equation 7 under “Experimental Procedures”) with the following parameters averaged for two titrations: K_1,obs = (7.0 ± 6.1) × 10⁸ m⁻¹, ΔH_1,obs = 11.6 ± 0.6 kcal/mol, K_2,obs = (4.3 ± 4.7) × 10⁵ m⁻¹, ΔH_2,obs = −14.1 ± 8.2 kcal/mol. C, 40 °C ITC titration of wtSSB (■) (1.1 μm) with (dA)₃₅ (32 μm). The smooth curve represents the best fit of the experimental isotherm to Equations 5 and 6 (“Experimental Procedures”) with the following parameters: n = 0.99 ± 0.01, K_obs = (3.4 ± 0.3) × 10⁷ m⁻¹ and ΔH_obs = −10.8 ± 0.1 kcal/mol. D, 40 °C, ITC titration of SSBΔC8 (●) (1.0 μm) with (dA)₃₅ (32 μm). The smooth curve represents the best fit of the experimental isotherm to Equations 5 and 6 (“Experimental Procedures”) with the following parameters: n = 1.02 ± 0.01, K_obs = (5.5 ± 2.0) × 10⁸ m⁻¹, and ΔH_obs = −11.9 ± 0.1 kcal/mol. The errors are shown as a S.D. Open symbols represent reference titrations of (dA)₃₅ in to the buffer.

FIGURE 4.

Thermodynamics of the binding of (dA)₃₅ to the first site on wtSSB and SSBΔC8. The values of K_1,obs and ΔH_1,obs were determined from analysis of the isotherms shown in Fig. 3 and supplemental Fig. S1. A, temperature dependence of log K_1,obs for wtSSB (■) and SSBΔC8 (□). B, the dependences of binding enthalpy (ΔH_1,obs) on temperature for wtSSB (●) and SSBΔC8 (○) (ΔCp = −0.89 ± 0.05 kcal/K mol and −0.93 ± 0.09 kcal/K mol, were obtained from a linear least squares fit of the data). The error bars are shown as S.D.

FIGURE 5.

Model for how the intrinsically disordered C-terminal tails inhibit binding of ssDNA to the SSB tetramer. In the absence of ssDNA (dark yellow), we hypothesize that the acid ends of the C-terminal tails interact with the positively charged ssDNA binding sites within the SSB core. Upon ssDNA binding, the C-terminal tails are displaced and made more accessible for interactions with other proteins.