Aromatic residues in the oligonucleotide binding domain are essential to the function of the single-stranded DNA binding protein of Helicobacter pylori.

Single-stranded DNA-binding protein (SSB) is essential to DNA replication, DNA repair, and homologous genetic recombination. Our previous study on the crystal structure of a C-terminally truncated SSB from Helicobacter pylori, HpSSBc, in complex with single-stranded DNA (ssDNA) suggests that several aromatic residues, including Phe37, Phe50, Phe56, and Trp84, were involved in ssDNA binding. To investigate the importance of these aromatic residues, the binding activity of four site-directed HpSSB mutants, including F37A HpSSB, F50A HpSSB, F56A HpSSB, and W84A HpSSB, was compared to that of wild-type HpSSB and HpSSBc by means of electrophoresis mobility shift assay (EMSA), tryptophan quenching fluorescence titration, and surface plasmon resonance (SPR). Molecular docking and molecular dynamic (MD) simulation of a F37A and a quadruple mutation model of HpSSBc support that the ssDNA-HpSSBc complex was destabilized when either one or four of the aromatic residues were mutated. The findings of this study suggest that mutation of the phenylalanine and tryptophan residues within the oligonucleotide-binding domain significantly diminished the ssDNA binding capability of HpSSB, highlighting the crucial role these aromatic residues play in the binding of ssDNA by HpSSB.