Thermodynamic and structural basis for relaxation of specificity in protein-DNA recognition.

As a novel approach to the structural and functional properties that give rise to extremely stringent sequence specificity in protein-DNA interactions, we have exploited "promiscuous" mutants of EcoRI endonuclease to study the detailed mechanism by which changes in a protein can relax specificity. The A138T promiscuous mutant protein binds more tightly to the cognate GAATTC site than does wild-type EcoRI yet displays relaxed specificity deriving from tighter binding and faster cleavage at EcoRI* sites (one incorrect base pair). AAATTC EcoRI* sites are cleaved by A138T up to 170-fold faster than by wild-type enzyme if the site is abutted by a 5'-purine-pyrimidine (5'-RY) motif. When wild-type protein binds to an EcoRI* site, it forms structurally adapted complexes with thermodynamic parameters of binding that differ markedly from those of specific complexes. By contrast, we show that A138T complexes with 5'-RY-flanked AAATTC sites are virtually indistinguishable from wild-type-specific complexes with respect to the heat capacity change upon binding (∆C°P), the change in excluded macromolecular volume upon association, and contacts to the phosphate backbone. While the preference for the 5'-RY motif implicates contacts to flanking bases as important for relaxed specificity, local effects are not sufficient to explain the large differences in ∆C°P and excluded volume, as these parameters report on global features of the complex. Our findings therefore support the view that specificity does not derive from the additive effects of individual interactions but rather from a set of cooperative events that are uniquely associated with specific recognition.