Interplay between Site-Specific Mutations and Cyclic Nucleotides in Modulating DNA Recognition by Escherichia coli Cyclic AMP Receptor Protein,

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Abstract
Mutagenesis of various amino acids in Escherichia coli cyclic AMP receptor protein (CRP) has been shown to modulate protein compressibility and dynamics [Gekko et al. (2004) Biochemistry 43, 3844−3852]. Cooperativity of cAMP binding to CRP and the apparent DNA binding affinity are perturbed [Lin and Lee (2002) Biochemistry 41, 11857−11867]. The aim of this study is to explore the effects of mutation on the surface chemistry of CRP and to define the consequences of these changes in affecting specific DNA sequence recognition by CRP. Furthermore, the role of the interplay between mutation and specific identity of the bound cyclic nucleotide in this DNA recognition was explored. In the current study, effects of eight site-specific mutations (K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L) on DNA recognition of four sequences (Class I (site PI of lac), Class II (site PI of gal), and synthetic sequences that are hybrids of Classes I and II sites) modulated by three different cyclic nucleotides (cAMP, cCMP, and cGMP) were investigated. All mutations altered the surface chemistry of CRP as evidenced by the change in elution properties of these proteins from different matrixes. While T127L, S62F, K52N, and H159L exhibited unexpected behavior under combinations of specific experimental conditions, such as the identity of bound cyclic nucleotide and DNA sequence, in general, results showed that the affinities of CRP for DNA were sequence-dependent, increasing in the order of lacgal26 < gal26 < lac26 < gallac26 for all the mutants in the presence of 200 μM cAMP. The apparent association constants significantly increased in the order of no cyclic nucleotide ≈ cGMP < cCMP < cAMP for all the examined DNA sequences. Linear correlation between the ΔG for CRP−DNA complex formation and the cooperativity energy for cAMP binding was observed with gallac26, gal26, and lacgal26; however, the slope of this linear correlation is DNA sequence dependent. Structural information was presented to rationalize the interplay between CRP sequence and cyclic nucleotides in defining the recognition of DNA sequences.