Origins of DNA-binding specificity: Role of protein contacts with the DNA backbone

A central question in protein-DNA recognition is the origin of the specific ity that permits binding to the correct site in the presence of excess, non specific DNA, In the P22 Are repressor, the Phe-10 side chain is part of th e hydrophobic core of the free protein but rotates out to pack against the sugar-phosphate backbone of the DNA in the repressor operator complex. Char acterization of a library of position 10 variants reveals that Phe is the o nly residue that results in fully active Are. One class of mutants folds st ably but binds operator with reduced affinity; another class is unstable. F V10, one member of the first class, binds operator DNA and nonoperator DNA almost equally well. The affinity differences between FV10 and wild type in dicate that each Phe-10 side chain contributes 1.5-2.0 kcal to operator bin ding but less than 0.5 kcal/mol to nonoperator binding, demonstrating that contacts between Phe-10 and the operator DNA backbone contribute to binding specificity. This appears to be a direct contribution as the crystal struc ture of the FV10 dimer is similar to wild type and the Phe-10-DNA backbone interactions are the only contacts perturbed in the cocrystal structure of the FV10-operator complex.