Intramolecular binding mode of the C-terminus ofEscherichia colisingle-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy

Abstract

Single-stranded DNA (ssDNA) binding protein (SSB) is an essential protein to protect ssDNA and recruit specific ssDNA-processing proteins. Escherichia coli SSB forms a tetramer at neutral pH, comprising a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain) of ∼64 amino acid residues. The C-terminal eight-residue segment of SSB (C-peptide) has been shown to interact with the OB-domain, but crystal structures failed to reveal any electron density of the C-peptide. Here we show that SSB forms a monomer at pH 3.4, which is suitable for studies by high-resolution nuclear magnetic resonance (NMR) spectroscopy. The OB-domain retains its 3D structure in the monomer, and the C-peptide is shown by nuclear Overhauser effects and lanthanide-induced pseudocontact shifts to bind to the OB-domain at a site that harbors ssDNA in the crystal structure of the SSB–ssDNA complex. 15N relaxation data demonstrate high flexibility of the polypeptide segment linking the C-peptide to the OB-domain and somewhat increased flexibility of the C-peptide compared with the OB-domain, suggesting that the C-peptide either retains high mobility in the bound state or is in a fast equilibrium with an unbound state.

The IDL of E. coli SSB links ssDNA and protein binding by mediating protein–protein interactions

“…This model has at its core, interactions between the acidic tip and OB‐folds of adjacent proteins. The current data show that this interaction is at its best, only transient . Furthermore, recent crystal structures of part of the acidic tip bound to interactome partners reveal that the tip does not bind to OB‐folds but instead binds to a hydrophobic pocket that contains a prominent basic residue …”

“…The current data show that this interaction is at its best, only transient. 29,30 Furthermore, recent crystal structures of part of the acidic tip bound to interactome partners reveal that the tip does not bind to OB-folds but instead binds to a hydrophobic pocket that contains a prominent basic residue. [26][27][28]31 To begin to understand how the linker region of SSB may link the seemingly disparate activities of cooperative ssDNA and protein binding, a combinatorial strategy was employed.…”

The tale of SSB

“…As ssDNA and C-peptide binding by the OB-fold are competitive, this model proposed that in solution, the C-termini of SSB are bound to the tetramer and are released upon ssDNA binding, making them available for interactions with target proteins. At low pH, the SSB tetramer dissociates into monomers and this was taken advantage of to show direct binding between the C-peptide and valines 29 and 58 of the OB-fold (Shishmarev et al, 2014). While this region interacts with the N-terminal domain when SSB is monomeric, this interaction is transient in the tetramer.…”

Weak and Transient Protein Interactions Determined by Solid‐State NMR

“…[13c, 14] Bacterial SSBs bind to ssDNAinasequence-independent manner, [15] protecting it from degradation or annealing to complementary sequences,w hilst recruiting many other proteins to the ssDNA. [14] Each 177 amino acid subunit of the E. coli SSB ( Figure S1) is comprised of as tructured Nterminal ssDNA-binding (oligonucleotide binding,O B) domain (residues 1-115), followed by an unstructured [16] and generally poorly conserved C-domain. At the extreme Cterminus (SSB-Ct, residues 170-177) is ah ighly conserved acidic motif (DFDDDIPF) [17] that is the site for the interaction of SSB with most of its partner proteins,i ncluding those in the replisome.…”

“…[19] Information concerning the C-terminal domain, however, is at lower resolution, and no electron density has been reliably observed beyond residue Gly114. [16,20] Nevertheless,the Ct motif and the OB domain interact in an autoinhibitory manner.T he affinity of SSB for ssDNAi s increased in an SSBDCt mutant, [14,21] and binding of the Ct to accessory proteins is inhibited by the OB domain. An isolated Ct peptide has higher affinity for the PriA helicase and the c subunit of DNAp olymerase III than the complete SSB molecule,and high affinity is restored when SSB is bound to ssDNA.…”