Cellular stability is constantly compromised due to unstable ssDNA being generated during DNA damage events and a variety of DNA metabolic processes. A group of proteins, known as single stranded DNA binding (SSB) proteins from the oligonucleotide binding (OB) domain family, are responsible for binding exposed ssDNA and thus providing temporary protection in these events.

Here, we present the high-resolution NMR structure of the SSB from the hyperthermophilic archaeon S. solfataricus (SsoSSB) in complex with a 6T ssDNA oligonucleotide. We have used a combination of intermolecular NOEs, paramagnetic resonance enhancement (PRE) data and mutagenesis to determine the structural basis of ssDNA recognition by SsoSSB. The majority of intermolecular NOEs were observed between three aromatic residues located in the hydrophobic binding cleft of SsoSSB and three of the six thymines resulting in a base-stacking binding mechanism. The complex structure is further stabilized by hydrophobic contacts made by two isoleucines and one threonine to several thymine methyl groups as well as several electrostatic interactions between positively charged lysines and negatively charged DNA side chain or backbone atoms. Notably, although SsoSSB binding to ssDNA is not sequence-specific, we were able to show a specific binding polarity using our PRE data. These data shed light on the molecular mechanism by which these SSBs interact with ssDNA in various DNA transactions.