Bacteriophage T4 gene 32 protein: Insights into its interaction with ssDNA, binding cooperativity, and conformational...

The single-stranded DNA binding protein of bacteriophage T4, gp32, has important roles in replication, recombination, and repair. gp32 possesses three domains: the central (core) domain which contains the binding trough for single-stranded DNA, the N-terminal domain, which interacts with the core domain of an adjacent ssDNA-bound protein, bringing about binding cooperativity, and the C-terminal domain, which interacts with other proteins involved in replication, recombination, and repair. The essential residues within the N-domain for the association with the adjacent DNA bound gp32, Lys-Arg-Lys-Ser-Thr, the "LAST Motif", is almost identical to the ssDNA-interactive residues within the core domain binding trough, and was the basis of a model in which a "closed" ⇄ "open" conformational change within core domain controls DNA binding. In this study, we show that alteration of the core domain LAST sequence, while maintaining its composition, can have an effect on the binding parameters, and may be the result of a shift in the closed-open equilibrium. Additionally, utilizing a gp32 truncated at residue 227, as well as amino acid substituted variants, we have further localized the residues within the core domain responsible for the protein-protein association leading to cooperative ssDNA binding. Truncation leads to an increase in the non-cooperative affinity for single-stranded nucleic acids, which can be explained by the absence of a closed conformation in this variant. The truncated protein forms a tight complex with core domain on a 12-residue oligonucleotide, a potential candidate for further structural study.