Characterization of an SSB-dT25 complex: structural insights into the S-shaped ssDNA binding conformation

Single-stranded DNA (ssDNA)-binding proteins (SSBs) play an important role in all DNA-dependent cellular processes, such as DNA replication, recombination, repair, and replication restart. The N-terminal domain of SSBs forms an oligonucleotide/oligosaccharide-binding (OB) fold for ssDNA binding. The SSB–dC35 complex structure has revealed how an Escherichia coli SSB (EcSSB) tetramer binds to 65-nucleotide (nt)-long ssDNA, namely, the (SSB)65 binding mode. Knowledge on whether the ssDNA-binding mode for EcSSB is typical for all SSBs or is bacterial strain and length dependent is limited. Here, we studied the ssDNA-binding properties of a Pseudomonas aeruginosa SSB (PaSSB) and investigated its interaction mode through crystallographic analysis. The complex crystal structure containing a PaSSB tetramer with two ssDNA chains was solved at a resolution of 1.91 A (PDB entry 6IRQ). Results revealed that each bound ssDNA dT25 adopts an S-shaped conformation. This binding mode, as shown by the complex structure of PaSSB, differs significantly from (SSB)65. ssDNA-binding contributions from aromatic residues in PaSSB, except the contribution of Trp54, were not significant. Using electrophoretic mobility shift analysis, we characterized the stoichiometry of PaSSB complexed with a series of ssDNA homopolymers. The minimal length of ssDNA required for PaSSB tetramer binding and the size of the ssDNA-binding site were 25 and 29 nt, respectively. These observations through structure–function analysis suggested that only two OB folds rather than four OB folds in PaSSB are enough for the formation of a stable complex with ssDNA. The PaSSB noninteracting OB folds proposed here may allow sliding via reptation in a dynamic ssDNA binding process.