Ability of E. coli Cyclic AMP Receptor Protein To Differentiate Cyclic Nucelotides:  Effects of Single Site Mutations

Abstract

Escherichia coli cyclic AMP receptor protein (CRP) is a global transcriptional regulator which controls the expression of many different genes. Although different cyclic nucleotides can bind to CRP with almost equal affinity, only in the presence of cAMP could wild-type CRP bind to specific DNA sequences. Molecular genetic studies have identified a class of mutants, CRP*, which either do not require exogenous cAMP for activation or can be activated by cGMP. Thus, these mutants might aid in identifying the structural elements that are involved in the modulation of CRP to correctly differentiate the messages embedded in cyclic nucleotides. In this in vitro study, five CRP* mutants, namely, D53H, S62F, G141Q, G141K, and L148R, were tested for their abilities to bind the lac promoter sequence and the effects of cyclic nucleotides in modulating DNA sequence recognition. For comparison, non-CRP* mutants K52N, T127L, H159L, and K52N/H159L were studied. cCMP and cGMP can replace cAMP as an allosteric effector in all of these CRP mutants except S62F and non-CRP* mutants. The D53H, G141Q, G141K, and L148R mutants exhibit significantly higher affinity for the lac promoter sequence than wild-type CRP while S62F and the non-CRP* mutants exhibit reduced affinity. To probe the pathway of communication, the energetics of subunit assembly in these mutants were monitored by sedimentation equilibrium, and the conformational states of these mutants were probed by proteolysis and accessibility of Cys178 to chemical modifications. Results from these studies imply that signals due to mutations are mostly transmitted through the subunit interface. Thus, residues in CRP outside of the cyclic nucleotide binding site modulate the ability of CRP to differentiate these three cyclic nucleotides through long-range communication. Furthermore, this study shows that CRP* mutations do not impart any unique properties to CRP except that the DNA binding constants are shifted to a regime of higher affinity.

Functional Roles of Loops 3 and 4 in the Cyclic Nucleotide Binding Domain of Cyclic AMP Receptor Protein from Escherichia coli

“…In recent studies, it was shown that a D53H mutation in loop 3 leads to enhancements of the magnitude of positive cooperativity in cAMP binding and affinity for specific DNA (21,22). These solution biophysical data are consistent with the proposal that loop 3 plays a role in interdomain and intersubunit communications, although the specific nature of this role is unknown.…”

“…Some point mutations, either within or just outside of these loops, have been reported to significantly affect the function of CRP, e.g. K52N, D53H, and S62F (beside loop 3) and E72A, K82Q, and S83K (beside loop 4) (21,22,(31)(32)(33)(34). Results from protein footprinting and NMR experiments indicate that the regions, including these loops exhibit significant environmental changes upon cAMP binding (18,19).…”

“…Furthermore, the nature of the perturbation could be manifested to yield different functional consequences. For example, mutations at residues 52 and 62 lead to decreases in both homotropic and heterotropic effects, but mutation at residues 53 leads to an opposite effect (21,22). Thus, apparently, perturbations of residues in loop 3 can modulate the allosteric effects either positively or negatively.…”

“…Protein Purification-Wild-type and mutant CRPs were purified from E. coli strain HMS174(DE3) using a previously described protocol (21,24). All purified CRP proteins were Ͼ99% homogeneous as judged by SDS-PAGE stained by Coomassie Blue; 50 -60 g of CRP was routinely loaded onto each lane.…”

“…At 230 M, the high affinity sites for cyclic nucleotides are occupied in all CRPs employed in this study (22). The detailed experimental and data analysis protocols have been previously described (21). Briefly, the data were fitted to the following equation by non-linear least-squares to determine the apparent association constant for CRP-DNA interaction, K,…”