Structural and energetic determinants of the DNA binding specificity of EcoRI endonuclease

Restriction endonuclease EcoRI is not only an invaluable tool in genetic engineering but also a paradigm for understanding the molecular mechanisms of protein-DNA recognition. EcoRI binds DNA of any sequence but strongly discriminates between its cognate GAATTC site and DNA of any other sequence.;A thermodynamic dissection of the binding free energy of the enzyme to DNA of various sequences has been performed. This analysis reveals that the heat capacity change (ΔCop) associated with the complex formation is the thermodynamic parameter that directly correlates with the preference of the protein for a particular DNA site. The large negative ΔC op accompanying site-specific binding is most likely a reflection of a cooperative molecular association process leading to the formation of a highly complementary protein-DNA interface.;The structural results reported in this study support this interpretation. The protein and DNA molecules undergo mutual conformational adaptations in order to achieve the sterical and chemical complementarity observed in the high resolution crystal structure of the specific complex. The crystal structure of the apo - EcoRI enzyme reported here at 2.7 Å resolution indicates that the unbound protein is characterized by uneven distribution of structural stability and that a major localized folding transition is coupled to site specific binding. The correlation of these findings with previous biophysical, genetic and thermodynamic studies of this system is discussed.;One of the well characterized but poorly understood aspects of the DNA binding specificity of EcoRI is the sensitivity of the enzyme to the identity of the DNA base-pairs flanking its cognate site. The first structural comparison between specific EcoRI - DNA complexes with two contexts variants reported here, reveals perfectly conserved recognition interactions within the 6 base-pair cognate site and subtle differences in the water-mediated contacts of the protein with the flanking bases. Our analysis suggests that changes in the dynamics of the water-mediated interactions outside the cognate site alters the local dynamics of the interface. Based on a better understanding of the factors that control the sensitivity of the enzyme to the sequence context of the target site, strategies for engineering protein variants with expanded site specificity could be designed.