Cellular stability is constantly compromised due to unstable ssDNA being generated during a variety of DNA metabolic processes. A group of proteins, known as single stranded DNA binding (SSB) proteins, 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 hyperthermophylic archeal organism Sulfolobus 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. Notable, although SsoSSB binding to ssDNA is not sequence-specific, we were able to show directionality using our PRE data.
Our structure also reveals similarities to the existing structure of human replication protein A (RPA) indicating that the molecular details of DNA-recognition of these SSBs are conserved throughout evolution.