Interactive and Dominant Effects of Residues 128 and 141 on Cyclic Nucleotide and DNA Bindings in Escherichia coli cAMP Receptor Protein

Abstract

The molecular events in the cAMP-induced allosteric activation of cAMP receptor protein (CRP) involve interfacial communications between subunits and domains. However, the roles of intersubunit and interdomain interactions in defining the selectivity of cAMP against other cyclic nucleotides and cooperativity in ligand binding are still not known. Natural occurring CRP mutants with different phenotypes were employed to address these issues. Thermodynamic analyses of subunit association, protein stability, and cAMP and DNA binding as well as conformational studies of the mutants and wild-type CRPs lead to an identification of the apparently dominant roles of residues 128 and 141 in the cAMP-modulated DNA binding activity of CRP. Serine 128 and the C-helix were implicated as playing a critical role in modulating negative cooperativity of cyclic nucleotide binding. A correlation was established between a weak affinity for subunit assembly and the relaxation of cyclic nucleotide selectivity in the G141Q and S128A/ G141Q mutants. These results imply that intersubunit interaction is important for cyclic nucleotide discrimination in CRP. The double mutant S128A/G141Q, constructed from two single mutations of S128A and G141Q, which exhibit opposite phenotypic characteristics of CRP ؊ and CRP * , respectively, assumes a CRP * phenotype and has biochemical properties similar to those of the G141Q mutant. These observations suggest that mutation G141Q exerts a dominant effect over mutation S128A and that the subunit realignment induced by the G141Q mutation can override the local structural disruption created by mutation S128A.

Analysis of the L116K Variant of CooA, the Heme-containing CO Sensor, Suggests the Presence of an Unusual Heme Ligand Resulting in Novel Activity

“…Importantly, the comparison of the structure of effector (CO)-free CooA with that of effector (cAMP)-bound CRP (6) reveals a repositioning of these two C-helices with respect to each other, suggesting a role of this repositioning in the activation of these proteins in response to their respective effectors. Consistent with this concept, alterations of particular amino acids within the C-helices exert a variety of effects on the activity in CRP (12,13) and in the fumarate and nitrate reductase activator protein (14), another member of this transcriptional activator family. It is therefore our working hypothesis that CO binding to the heme of CooA alters the positioning of the C-helices and that this repositioning effectively transmits the effector-binding signal through the protein and leads to the reorganization of the DNA-binding domains.…”

cAMP Receptor Protein from <i>Escherichia coli</i> as a Model of Signal Transduction in Proteins – A Review

“…On the other hand, the substitution of glycine in position 141 (with glutamine) causes the loss of CRP's ability to discriminate between cAMP and other cyclic nucleotides, although Gly141 is not involved in the interaction with cAMP and in the interaction between subunits [Cheng and Ching Lee, 1998]. …”

Redox-mediated Transcriptional Activation in a CooA Variant

“…Similar to that of effector-bound CRP, the structure of effector-free CooA indicated that a single ␣-helix (designated the C-helix) in each monomer serves to create the intersubunit dimerization domain (18). In CRP and FNR, alterations of particular amino acids in the center of this helix have a variety of effects on activity (25)(26)(27). In this report, we have identified CooA variants altered in the C-helix that have a perturbed ligation structure in the oxidized (Fe III ) form.…”