THERMODYNAMICS OF SPECIFIC AND NONSPECIFIC DNA-BINDING BY THE C-MYB DNA-BINDING DOMAIN

The thermodynamics of the c-Myb DNA-binding domain (R2R3) interaction with its target DNA have been analyzed using isothermal titration calo rimetry and amino acid mutagenesis. The enthalpy of association betwee n the standard R2R3, the Cys130 mutant substituted with Ile, and the c ognate DNA is -12.5 (+/-0.1) kcal mol(-1) at pH 7.5 and at 20 degrees C, and this interaction is enthalpically driven throughout the physiol ogical temperature range. In order to understand the DNA recognition m echanism, several pairs of interactions were investigated using single and multiple-base alterations with single and multiple-amino acid sub stituted mutants. The interactions between the standard R2R3 and many non-cognate DNAs were accompanied by binding enthalpy changes and heat capacity changes, although their affinities were reduced. The roles o f the electrostatic interactions in binding to the cognate and the non -cognate DNAs were also analyzed from the dependency of the thermodyna mic parameters on the salt concentration. The heat capacity change was found to be significantly dependent upon the salt concentration. Seve ral mutant proteins bound to the multiple-base altered DNA with very s mall enthalpy changes, although they bound to the cognate and the sing le-base altered DNAs with detectable enthalpy and heat capacity change s. From the thermodynamic cycles derived from the DNA binding of the a mino acid substituted R2R3 to the base substituted DNA duplexes, the i ndividual thermodynamic mechanisms of the specific DNA recognition of R2R3 were dissected. The local folding mechanism was highlighted by th e substitution of Pro with either Gly or Ala at the Linker between R2 and R3. The characteristic thermodynamic features of specific and nons pecific DNA binding are discussed. (C) 1998 Academic Press Limited.