Identification of the SSB binding site on E. coli RecQ reveals a conserved surface for binding SSB's C terminus

Abstract

RecQ DNA helicases act in conjunction with heterologous partner proteins to catalyze DNA metabolic activities, including recombination initiation and stalled replication fork processing. For the prototypical Escherichia coli RecQ protein, direct interaction with single-stranded DNA-binding protein (SSB) stimulates its DNA unwinding activity. Complex formation between RecQ and SSB is mediated by the RecQ winged-helix domain, which binds the nine C-terminal-most residues of SSB, a highly conserved sequence known as the SSB-Ct element. Using nuclear magnetic resonance and mutational analyses, we identify the SSB-Ct binding pocket on E. coli RecQ. The binding site shares a striking electrostatic similarity with the previously identified SSB-Ct binding site on E. coli exonuclease I, although the SSB binding domains in the two proteins are not otherwise related structurally. Substitutions that alter RecQ residues implicated in SSB-Ct binding impair RecQ binding to SSB and SSB/DNA nucleoprotein complexes. These substitutions also diminish SSB-stimulated DNA helicase activity in the variants, although additional biochemical changes in the RecQ variants indicate a role for the winged-helix domain in helicase activity beyond SSB protein binding. Sequence changes in the SSB-Ct element are sufficient to abolish interaction with RecQ in the absence of DNA and to diminish RecQ binding and helicase activity on SSB/DNA substrates. These results support a model in which RecQ has evolved an SSB-Ct binding site on its winged-helix domain as an adaptation that aids its cellular functions on SSB/DNA nucleoprotein substrates.

Figure 1. Structural basis of RecQ SSB-Ct binding

(A) Schematic domain diagram of E. coli RecQ and SSB proteins. Domains in RecQ (Helicase, Zinc-binding (Zn), winged-helix (WH, green), and Helicase and RNase D C-terminal (HRDC)) and SSB (OB) are labeled. The sequence of the residues from SSB comprising the SSB-Ct element is given. An arrow indicates the elements of RecQ and SSB that are sufficient for complex formation. (B) ¹⁵N HSQC spectral overlay of the RecQ-WH domain (black) and a 1:1 mixture of the RecQ-WH domain and SSB-Ct peptide (red). The largest 10 chemical shift differences between the spectra are labeled. (C) Histogram of the difference in amide proton and nitrogen resonance chemical shifts of the RecQ-WH with and without the addition of a 1:1 mixture of the SSB-Ct peptide. The largest differences (>0.3 ppm) are labeled and colored in magenta on secondary structural elements (helices as boxes, strands as arrows) of the RecQ-WH domain in the context of the crystal structure of the RecQ catalytic core . (D) Structure and electrostatics of the putative RecQ SSB-Ct binding site. (left) The ten RecQ-WH domain residues with the highest chemical shift differences upon SSB-Ct binding are labeled and colored in magenta on a ribbon diagram of the RecQ catalytic core structure. (right) Electrostatic representation of the surface of the RecQ catalytic core (red, blue and white for negative, positive and neutral, respectively) is shown in the same perspective as the ribbon diagram. Residues in addition to those identified by NMR that were altered in biochemical studies are labeled in the electrostatic diagram.

Figure 2. RecQ variant SSB binding to SSB

(A) Ammonium sulfate co-precipitation pellets of RecQ variant and SSB mixtures were resolved by PAGE (top). Quantitation of the intensity of the RecQ variant band relative to wild-type protein is shown below the gel. Values are the mean of three measurements and one standard deviation as the error. (B) Alanine-substituted residues with modest (yellow) and severe (red) defects in SSB co-precipitation are shown on a ribbon diagram of the RecQ catalytic core structure.

Figure 3. RecQ variants DNA and SSB/DNA binding

Electrophoretic mobility shift analysis of RecQ variant proteins binding to partial duplex DNA is shown in the absence (left, each panel) and presence (right, each panel) of SSB.

Figure 4. Effects of SSB-Ct sequence changes on RecQ binding

(A) Ammonium sulfate co-precipitation pellets of RecQ and SSB variant mixtures were resolved by PAGE. (B) Electrophoretic mobility shift analysis of RecQ binding to partial duplex DNA prebound by SSB protein variants. (C) DNA unwinding by wild type RecQ in the absence and presence of SSB and SSB proteins variants.

Figure 5

Comparison of the SSB-Ct binding site from Exonuclease I (left) , and RecQ (right). Surface representations of each molecule are colored by electrostatic features (red, blue and white for negative, positive and neutral, respectively). The SSB-Ct peptide is shown in orange on the Exonuclease I structure. The basic ridge, the arginine at the lip of the binding pocket, and the hydrophobic pocket are highlighted in both images.