Dissecting the Functional Domains of Phi11 Gp13 through Deletion Mutagenesis.

Single-stranded DNA-binding proteins (SSBs) are found across all life domains, including viruses. They stabilize transient single-stranded DNA (ssDNA) intermediates, vital for nucleic acid metabolism. While bacterial SSBs typically form homotetramers with an N-terminal oligonucleotide/oligosaccharide binding (OB) fold, the bacteriophage Phi11 Gp13 is a homodimer with a C-terminal OB-fold. This study investigates functional domains in Phi11 Gp13 via biochemical and biophysical characterization of N and C-terminal deletion mutants. Specific N-terminal (residues 2-8) and C-terminal (residues 177-184) residues are critical for efficient ssDNA binding affinity. Notably, C-terminal (C1, C2) and N-terminal (N1) deletions resulted in a novel shift in binding site size from 25 nucleotides (for Gp13) to 20 nucleotides, suggesting peripheral residues impose structural constraints on the DNA-binding domain. On the contrary, further N-terminal deletions, as seen in case of N5, completely abolished DNA binding, highlighting the importance of residues R6-E12 in forming the DNA-binding surface. Despite exhibiting differential ssDNA binding, most mutants retained their dimeric state, suggesting the dimerization domain lies outside the terminal regions of the protein. While overall structural integrity was largely preserved, localized secondary and tertiary changes (e.g., increased alpha-helical content, subtle red shifts in tryptophan fluorescence, destabilizations in C2 and N5) were observed, which correlated directly with ssDNA binding affinity. Furthermore, Gp13 interacted with Staphylococcus aureus ( S. aureus) RecA. This research reveals the important links between the primary amino acid sequence, its three-dimensional conformation, and the biological function of Gp13.