Effect of mutations at the monomer-monomer interface of cAMP receptor protein on specific DNA binding.

To determine the thermodynamic role of binding of an operon to cAMP receptor protein (CRP) in the activation of transcription, isothermal titration calorimetry measurements were performed on the binding of three 40-base pair DNA sequences to the cyclic nucleoside complexes of CRP and its mutants at 296 K. The three 40-base pair sequences consisted of a consensus DNA (conDNA) duplex derived from the CRP-binding site sequences of the operons activated by CRP and two DNA sequences based on the CRP-binding site sequences of the lac operon (lacDNA) and of the gal operon (galDNA). The mutants of CRP consisted of a T127L mutant, a S128A mutant, and a mutant containing both mutations (CRP*) which not only alter the transcriptional activity of the CRP complexes but also are involved in the monomer-monomer interfacial interactions of the CRP dimer. The binding reactions of the DNA duplexes to the fully cNMP-ligated CRP-mutant complexes were endothermic with binding constants as high as 6.6 +/- 1.1 x 10(6) M-1 (conDNA.CRP(cAMP)2). ConDNA binding to the unligated T127L and CRP* mutants was observed as well as conDNA and lacDNA binding to CRP with cAMP bound to only one monomer. The reduction of the binding constants with increase in KCl concentration indicated the formation of two ion pairs for the cAMP-ligated CRP and S128A complexes and four ion pairs for the cAMP-ligated T127L and CRP* complexes. Reduction of the DNA binding constants upon substitution of D2O for H2O in the buffer, the large heat capacity changes, and the enthalpy-entropy compensation exhibited by the binding reactions indicate the importance of dehydration in the binding reaction. Small angle neutron scattering measurements on the lacDNA.CRP(cAMP)2 complex in D2O/H2O mixtures show that the DNA is bent around the cAMP-ligated protein in solution.