Differential perturbation of intersubunit and interdomain communications by glycine 141 mutation in Escherichia coli CRP

Upon binding of cAMP, concomitant changes in CRP structure across the subunit and domain interfaces are observed. In order to identify the structural elements involved in the coupling of interfacial interactions, structural perturbation was introduced at residue 141 by site-directed mutagenesis. Thermodynamic parameters defining protein stability, cAMP binding, and subunit assembly of the mutant were determined. Conformational changes probed by proteolytic digestion and fluorescence signal reported by the fluorescein-labeled C178 lead to a dissection of the contribution of the intersubunit and interdomain interactions, respectively, in the cAMP- modulated DNA binding of CRP. In the absence of cAMP, mutant G141Q is sensitive to protease attack at the subunit interface, an established property of wild type CRP observed only in the presence of cAMP. Although the G141Q mutant assumes a subunit alignment similar to that of the activated CRP, this mutant absolutely requires cyclic nucleotide for specific DNA interaction. Monitoring the fluorescence probe attached to the C-terminal DNA binding domain of the G141Q mutant showed that the DNA binding domain responds quantitatively to the binding of cyclic nucleotide to the N-terminal domain. This result suggests that domain reorientation is a required structural change in addition to subunit alignment. In summary, mutation at G141 has differentially perturbed the communication network which involves the interfacial interactions between subunits and domains. The G141Q CRP mutant assumes a conformation that partially resembles the active form represented by the observed subunit realignment, but complete activation of the mutant requires binding of cyclic nucleotide which induces the reorientation of domains. Furthermore, the G → Q mutation leads to a loss in the discriminatory power of CRP for only cAMP. Other cyclic nucleotides are capable of activating this mutant.