Bound or Free: Interaction of the C-Terminal Domain of Escherichia coli Single-Stranded DNA-Binding Protein (SSB) with the Tetrameric Core of SSB

Abstract

Single-stranded DNA (ssDNA)-binding protein (SSB) protects ssDNA from degradation and recruits other proteins for DNA replication and repair. Escherichia coli SSB is the prototypical eubacterial SSB in a family of tetrameric SSBs. It consists of a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain). The eight-residue C-terminal segment of SSB (C-peptide) mediates the binding of SSB to many different SSB-binding proteins. Previously published nuclear magnetic resonance (NMR) data of the monomeric state at pH 3.4 showed that the C-peptide binds to the OB-domain at a site that overlaps with the ssDNA binding site, but investigating the protein at neutral pH is difficult because of the high molecular mass and limited solubility of the tetramer. Here we show that the C-domain is highly mobile in the SSB tetramer at neutral pH and that binding of the C-peptide to the OB-domain is so weak that most of the C-peptides are unbound even in the absence of ssDNA. We address the problem of determining intramolecular binding affinities in the situation of fast exchange between two states, one of which cannot be observed by NMR and cannot be fully populated. The results were confirmed by electron paramagnetic resonance spectroscopy and microscale thermophoresis. The C-peptide-OB-domain interaction is shown to be driven primarily by electrostatic interactions, so that binding of 1 equiv of (dT)35 releases practically all C-peptides from the OB-domain tetramer. The interaction is much more sensitive to NaCl than to potassium glutamate, which is the usual osmolyte in E. coli. As the C-peptide is predominantly in the unbound state irrespective of the presence of ssDNA, long-range electrostatic effects from the C-peptide may contribute more to regulating the activity of SSB than any engagement of the C-peptide by the OB-domain.

The tale of SSB

“…These interactions are critical to cooperative binding of ssDNA as well as to SSB interactome function. In addition, and consistent with the work of others, the model proposes that the acidic tip is not involved in protein binding per se but is instead a regulatory element (Mason et al, 2013; Su et al, 2014). …”

“…When the protein is in solution, the IDL adopts a variety of conformations with the acidic tip making only transient interactions with the N-terminal core (Matsumoto et al, 2000; Savvides et al, 2004; Su et al, 2014). The presence of the acidic tip is critical as it may prevent an IDL from binding to the tetramer from which it emanates.…”

“…The inherent flexibility in this region of the protein as suggested by our sequence analysis may then facilitate sliding of the second tetramer relative to the first so that they occupy immediately adjacent positions on the ssDNA. There is evidence for tetramer sliding on ssDNA (Su et al, 2014). Continued IDL-OB-fold interactions would then facilitate rapid and complete binding of the ssDNA in highly cooperative fashion.…”

“…In fact, in solution, the acidic tip spends the majority of its time away from the tetramer core making itself available for protein-protein interactions. (Su et al, 2014). This was confirmed in vivo where binding of SSB in the absence of DNA to the fork rescue helicases RecG and PriA was observed (Yu et al, 2016).…”

Glutamate promotes SSB protein–protein Interactions via intrinsically disordered regions

“…The C-terminal domain of SSB (residues 113–177) is not observable in any crystal structures, even when SSB is bound to ssDNA [30], suggesting that these C-terminal tails are intrinsically disordered, as first proposed based on its primary structure [31] and biochemical properties [32–34]. The acidic tip can interact with an unoccupied DNA binding site on SSB, which inhibits ssDNA binding [8, 34, 35].…”