Kinetic Studies of cAMP-induced Allosteric Changes in Mutants T127I, S128A, and T127I/S128A of the cAMP Receptor Protein from Escherichia coli

Abstract

The cAMP receptor protein (CRP) regulates the expression of several genes in Escherichia coli. The protein is a homodimer, and each monomer is folded into two distinct structural domains. After allosteric transitions resulting from the binding of cAMP, CRP specifically binds to DNA and activates transcription. We have used stopped-flow fluorometry measurements of CRP mutants bearing amino acid substitutions T127I, S128A, and T127I/S128A to study the kinetics of conformational changes in the protein induced by cAMP binding. Amino acid substitutions at positions 127 and 128 were chosen because these residues play a crucial role in interdomain and intersubunit communication during allosteric transition. Using N-iodoacetylaminoethyl-5-naphthylamine-1-sulfonic acid-labeled Cys 178 , localized in the protein helix-turn helix motif, we observed conformational changes in the helix-turn helix, localized in the C-terminal domain, upon binding of cAMP to high affinity sites (CRP⅐cAMP 2 ) in the N-terminal domain of CRP. The rate constants for the forward and backward conformational changes depend on the amino acid substitution: k c ‫؍‬ 3.62 s ؊1 and k ؊c ‫؍‬ 3.13 s ؊1 for CRP T127I and k c ‫؍‬ 0.42 s ؊1and k ؊c ‫؍‬ 0.78 s ؊1 for CRP S128A. These values can be compared with k c ‫؍‬ 9.7 s ؊1 and k ؊c ‫؍‬ 0.31 s ؊1 for wildtype CRP. The observed conformational changes can be described by the sequential model of allostery, with the amino acid substitutions influencing the allosteric changes. In the case of the double mutant, the observed rate constant of cAMP binding supports the suggestion that this unligated mutant possesses the structure that is close to the allosteric conformation necessary for promoter binding. The results of intrinsic fluorescence measurements suggest that the formation of the CRP⅐cAMP 4 complex results from displacement of equilibrium between the two forms of the CRP⅐cAMP 2 complex in the mutants studied, similar to wild-type CRP. The observed conformational changes occur according to a concerted model of allostery, and isomerization equilibrium between the two CRP states depends on the amino acid substitution. The data presented in this study indicate that Ser 128 and Thr 127 in CRP play an important role in the kinetics of intramolecular transitions triggered by cAMP.The cAMP receptor protein (CRP) 1 regulates the expression of Ͼ100 genes in Escherichia coli. CRP is a dimeric protein composed of two identical subunits of 209 amino acid residues with a molecular mass of 22.6 kDa (1, 2). Each subunit is folded into two distinct domains (3). The larger N-terminal domain contains a binding site for cAMP in the anti-conformation, and the smaller C-terminal domain contains a helix-turn-helix (HTH) motif responsible for DNA recognition and binding. The two domains are connected with a short hinge region composed of residues 135-138. Crystal structure studies have shown that the protein additionally possesses a second binding site (located between the hinge and the turn of HTH) that binds cAMP in the syn-co...

Kinetic studies of cAMP-induced propagation of the allosteric signal in the cAMP receptor protein from Escherichia coli with the use of site-directed mutagenesis

“…Thus, the data are consistent with our previous kinetic studies using wt CRP [21], CRP T127I and CRP S128A mutants [22], suggesting that the observed change in the fluorescence intensity of labeled proteins resulted from a conformational change of the double ligated protein. Therefore, the obtained data in the present study were also analyzed in terms of a sequential (KNF) model of allostery, represented by Scheme 2, where K 1 and K 2 are intrinsic dissociation constants for the binding of the first and the second molecule, respectively; k c and k −c are the rate constants describing the conformational change, CRP and CRP' represent the protein before and after that process, respectively.…”

“…Because the estimated value for the wt CRP is low and equals to 0.032, one can expect that almost all double ligated wt CRP would exist in the active conformation [21]. Similarly to the mutation introduced at positions 127 and 128 [22], the amino acid substitution at position 138 can also shift the equilibrium between the two conformational forms of CRP towards the low or high activity conformation ( Table 3). The affinity can be characterized by the dissociation constants of the considered mutants from the promoter region, which was determined for the lac operon promoter.…”

“…Conformational changes observed at mM concentrations were described by a concerted (MWC) model. In general, the observed conformational changes clearly indicated long-range structural communication between the N-terminal and C-terminal domains of CRP remaining under kinetics control [21,22].…”

“…Our previous stopped-flow studies investigating the kinetics of conformational changes induced by cAMP binding to wt CRP and its several mutants, showed that the allosteric changes induced by M concentrations of cAMP occur in a one-step reaction that takes place according to a sequential (KNF) model [21,22]. Conformational changes observed at mM concentrations were described by a concerted (MWC) model.…”

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

“…As a consequence, there is a significant reduction in the affinity of the binding sites localized between the protein domains, and the dissociation constant for these sites is about 8.5 m M . The parameters of cyclic AMP binding to both binding sites determined by kinetic experiments unambiguously show that the point mutation perturbs the communication between the protein domains [Polit et al, 2003b].…”