Wild-type cAMP Receptor Protein Research Articles

Two-State Allosteric Modeling Suggests Protein Equilibrium as an Integral Component for Cyclic AMP (cAMP) Specificity in the cAMP Receptor Protein ofEscherichia coli

Activation of the cAMP receptor protein (CRP) from Escherichia coli is highly specific to its allosteric ligand, cAMP. Ligands such as adenosine and cGMP, which are structurally similar to cAMP, fail to activate wild-type CRP. However, several cAMP-independent CRP variants (termed CRP*) exist that can be further activated by both adenosine and cGMP, as well as by cAMP. This has remained a puzzle because the substitutions in many of these CRP* variants lie far from the cAMP-binding pocket (>10 A) and therefore should not directly affect that pocket. Here we show a surprising similarity in the altered ligand specificity of four CRP* variants with a single substitution in D53S, G141K, A144T, or L148K, and we propose a common basis for this phenomenon. The increased active protein population caused by an equilibrium shift in these variants is hypothesized to preferentially stabilize ligand binding. This explanation is completely consistent with the cAMP specificity in the activation of wild-type CRP. The model also predicts that wild-type CRP should be activated even by the lower-affinity ligand, adenosine, which we experimentally confirmed. The study demonstrates that protein equilibrium is an integral factor for ligand specificity in an allosteric protein, in addition to the direct effects of ligand pocket residues.

Study of Highly Constitutively Active Mutants Suggests How cAMP Activates cAMP Receptor Protein

The cAMP receptor protein (CRP) of Escherichia coli undergoes a conformational change in response to cAMP binding that allows it to bind specific DNA sequences. Using an in vivo screening method following the simultaneous randomization of the codons at positions 127 and 128 (two C-helix residues of the protein interacting with cAMP), we have isolated a series of novel constitutively active CRP variants. Sequence analysis showed that this group of variants commonly possesses leucine or methionine at position 127 with a beta-branched amino acid at position 128. One specific variant, T127L/S128I CRP, showed extremely high cAMP-independent DNA binding affinity comparable with that of cAMP-bound wild-type CRP. Further biochemical analysis of this variant and others revealed that Leu(127) and Ile(128) have different roles in stabilizing the active conformation of CRP in the absence of cAMP. Leu(127) contributes to an improved leucine zipper at the dimer interface, leading to an altered intersubunit interaction in the C-helix region. In contrast, Ile(128) stabilizes the proper position of the beta4/beta5 loop by functionally communicating with Leu(61). By analogy, the results suggest two direct local effects of cAMP binding in the course of activating wild-type CRP: (i) C-helix repositioning through direct interaction with Thr(127) and Ser(128) and (ii) the concomitant reorientation of the beta4/beta5 loop. Finally, we also report that elevated expression of T127L/S128I CRP markedly perturbed E. coli growth even in the absence of cAMP, which suggests why comparably active variants have not been described previously.

Role of Residue 138 in the Interdomain Hinge Region in Transmitting Allosteric Signals for DNA Binding in Escherichia coli cAMP Receptor Protein

The cAMP receptor protein (CRP) of Escherichia coli is a transcription factor. The affinity of CRP for a specific DNA sequence is significantly enhanced as a consequence of the binding of the allosteric effector, cAMP. The hinge region, particularly residues 136 and 138, connecting the cAMP and DNA binding domains of CRP has been proposed to play essential roles in transmitting the allosteric signals. To probe the specific role of residue 138, eight D138 mutants and wild-type CRP were tested for their ability to bind the lac26 and gallac26 promoter sequences in this study. A correlation was established between DNA binding affinity and side chain solvation free energy, namely, an increasing specific DNA affinity with an increasing hydrophilicity of the side chain of residue 138. In addition, a linear correlation was found between DNA binding affinity and the energetics of subunit assembly. The ability of CRP to distinguish between cAMP and cGMP as an allosteric activator of DNA binding is weakened with higher energetics of subunit assembly. This correlation indicates that quaternary constraint leads to a constraint of the DNA binding domain. This observation is consistent with the concept that an optimum quaternary structural constraint is important in CRP exhibiting its allosteric properties. The stability of CRP was monitored by Trp fluorescence and circular dichroism in the presence of guanidine hydrochloride. These spectroscopic data revealed nonidentical denaturation profiles. Since the Trp residues are located exclusively in the beta-roll cyclic nucleotide binding domain, the denaturation profiles reveal the stability of the beta-roll structure. This study produces another linear correlation between DNA binding affinity in the presence of cAMP and cGMP and the stability of the beta-roll; namely, the stability of the beta-roll structure leads to a decrease in DNA binding affinity. All these correlations indicate the importance of structural stability and dynamics in the ability of CRP to manifest its intrinsic allosteric properties.

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

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 Cys178, 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 (CRP.cAMP2) in the N-terminal domain of CRP. The rate constants for the forward and backward conformational changes depend on the amino acid substitution: kc = 3.62 s-1 and k-c = 3.13s-1 for CRP T127I and kc = 0.42 s-1 and k-c = 0.78 s-1 for CRP S128A. These values can be compared with kc = 9.7 s-1 and k-c = 0.31 s-1 for wild-type 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 CRP.cAMP4 complex results from displacement of equilibrium between the two forms of the CRP.cAMP2 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 Ser128 and Thr127 in CRP play an important role in the kinetics of intramolecular transitions triggered by cAMP.

The effect of Ser 128 substitution on the structure and stability of cAMP receptor protein from Escherichia coli.

Kinetic measurements of denaturation and renaturation of two mutants of cAMP receptor protein (CRP) at position 128, namely Ser --> Ala and Ser --> Pro, were performed in order to assess changes introduced by the mutation in the quaternary structure and protein stability. No significant changes were found in the unfolding/refolding reactions. However, small perturbations in the dissociation of CRP dimer can be seen, which indicate that subunit interactions are influenced by the mutation. Studies of intrinsic fluorescence quenching of these two mutants are also reported, showing changes compared with wild-type protein. Near-UV circular dichroism measurements indicate, however, that Trp residues remain in the same environment as in the wild-type CRP. It is proposed that Ser at position 128 is involved in maintaining the proper domain alignment within CRP subunits.

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

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.

Effect of cAMP Binding Site Mutations on the Interaction of cAMP Receptor Protein with Cyclic Nucleoside Monophosphate Ligands and DNA

Although cAMP binding to wild type cAMP receptor protein (CRP) induces specific DNA binding and activates transcription, cyclic nucleoside monophosphate (cNMP) binding to the CRP mutant Ser128 --> Ala does not, whereas the double CRP mutant Thr127 --> Leu/Ser128 --> Ala activates transcription even in the absence of cNMP. Isothermal titration calorimetry measurements on the cNMP binding reactions to the S128A and T127L/S128A mutants show that the reactions are mainly entropically driven as is cAMP binding to CRP. In contrast to cAMP binding to CRP, the binding reactions are noncooperative and exothermic with binding enthalpies (DeltaHb) ranging from -23.4 +/- 0.9 kJ mol-1 for cAMP binding to S128A at 39 degrees C to -4.1 +/- 0.6 kJ mol-1 for cAMP binding to T127L/S128A at 24 degrees C and exhibit enthalpy-entropy compensation. To account for the inactivity of the S128A mutant, in vitro and in vivo DNA binding experiments were performed on the cAMP-ligated S128A mutant. The cAMP-ligated S128A mutant binds to the consensus DNA binding site with approximately the same affinity as that of cAMP-ligated CRP but forms a different type of complex, which may account for loss of transcriptional activity by the mutant. Energy minimization computations on the cAMP-ligated S128A mutant show that amino acid conformational differences between S128A and CRP occur at Ser179, Glu181, and Thr182 in the center of the DNA binding site, implying that these conformational changes may account for the difference in DNA binding.

Absolute requirement of cyclic nucleotide in the activation of the G141Q mutant cAMP receptor protein from Escherichia coli.

cAMP receptor protein (CRP), when interacting with cAMP, controls the expression of a network of catabolite-sensitive genes in Escherichia coli. To understand the molecular events that lead to the activation of CRP, a combined approach of site-directed mutagenesis and thermodynamic analysis was employed to study a member of a specific class of CRP mutant, CRP, which activates the in vivo expression of CRP-dependent operons in cya- strains in the absence of exogenous cAMP. Results from in vitro studies show that the CRP mutant G141Q absolutely requires cAMP for interacting with specific DNA. A quantitative comparison of the thermodynamic parameters governing ligand binding and DNA-protein complex formation in the presence of different cyclic nucleotides leads to the conclusion that this CRP mutant is activated only in the presence of cyclic nucleotides. The specificity toward cyclic nucleotides exhibited by wild-type CRP is lost in this mutant. Furthermore, the binding affinity of the ligand for the first binding site of the mutant is essentially the same as that of wild-type CRP regardless of the identity of the cyclic nucleotide. Hence, the observed in vivo activation of CRP-dependent operons by G141Q in the absence of exogenous cAMP is most likely the consequence of the replacement of cAMP by other cyclic nucleotides to activate the mutant. It is also possible that trace levels of cAMP present in the cya- strain could account for the in vivo activation of the mutant. Furthermore, these results indicate that this CRP mutant does not assume the activated conformation in the absence of cyclic nucleotides, in contrast to the current model derived from results of in vivo studies.

Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 83, 127 and 128 of the cyclic nucleotide binding pocket.

The cyclic 3', 5' adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute cysteine or glycine for serine 83; cysteine, glycine, isoleucine, or serine for threonine 127; and threonine or alanine for serine 128. Cells that expressed the binding pocket residue-substituted forms of CRP were characterized by measurements of beta-galactosidase activity. Purified wild-type and mutant CRP preparations were characterized by measurement of cAMP binding activity and by their capacity to support lacP activation in vitro. CRP structure was assessed by measurement of sensitivity to protease and DTNB-mediated subunit crosslinking. The results of this study show that cAMP interactions with serine 83, threonine 127 and serine 128 contribute to CRP activation and have little effect on cAMP binding. Amino acid substitutions that introduce hydrophobic amino acid side chain constituents at either position 127 or 128 decrease CRP discrimination of cAMP and cGMP. Finally, cAMP-induced CRP structural change(s) that occur in or near the CRP hinge region result from cAMP interaction with threonine 127; substitution of threonine 127 by cysteine, glycine, isoleucine, or serine produced forms of CRP that contained, independently of cAMP binding, structural changes similar to those of the wild-type CRP:cAMP complex.

Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 72 and 82 of the cyclic nucleotide binding pocket.

The 3', 5' cyclic adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute leucine, glutamine, or aspartate for glutamate 72; and lysine, histidine, leucine, isoleucine, or glutamine for arginine 82. Substitutions were made in wild-type CRP and in a CRP*, or cAMP-independent, form of the protein to assess the effects of the amino acid substitutions on CRP structure. Cells containing the binding pocket residue-substituted forms of CRP were characterized through beta-galactosidase activity and by measurement of cAMP binding activity. This study confirms a role for both glutamate 72 and arginine 82 in cAMP binding and activation of CRP. Glutamine or leucine substitution of glutamate 72 produced forms of CRP having low affinity for the cAMP and unresponsive to the nucleotide. Aspartate substituted for glutamate 72 produced a low affinity cAMP-responsive form of CRP. CRP has a stringent requirement for the positioning of the position 72 glutamate carboxyl group within the cyclic nucleotide binding pocket. Results of this study also indicate that there are differences in the binding requirements of cAMP and cGMP, a competitive inhibitor of cAMP binding to CRP.

Pivotal role of amino acid at position 138 in the allosteric hinge reorientation of cAMP receptor protein.

The cAMP receptor protein (CRP) of Escherichia coli needs cAMP for an allosteric change to regulate gene expression by binding to specific DNA sites. The hinge region connecting the DNA-binding domain to the cAMP-binding domain has been proposed to participate in the cAMP-induced allosteric change necessary to adjust C and D alpha-helices for movement of the DNA-binding F alpha-helix away from the protein surface. The role of the hinge region for a conformation change in CRP was tested by studying the effects of single amino acid substitutions at residue 138 located within the hinge. Physiological studies of wild-type and mutant cells and biochemical analysis of purified wild-type and mutant CRP revealed at least three groups of altered CRPs: (i) CRP that behaves like wild type (CRP+); (ii) CRP that binds cAMP but does not complete the structural changes required for specific DNA binding, proteolytic cleavage, and transcription activation (CRPallo); and (iii) CRP that shows some or all of these conformational changes without cAMP (CRP*). These results show a pivotal role of position 138 from which change emanates and provide further evidence that a hinge reorientation involving residue 138 is involved in the interhelical adjustments.

Single amino acid substitutions in the cAMP receptor protein specifically abolish regulation by the CytR repressor in Escherichia coli.

Promoters in Escherichia coli that are negatively regulated by the CytR repressor are also activated by the cAMP receptor protein (CRP) complexed to cAMP; as a characteristic, these promoters encode tandem binding sites for cAMP-CRP. In one such promoter, deoP2, CytR binds to the region between the tandem CRP binding sites with a relatively low affinity; in the presence of cAMP-CRP, however, the repressor and activator bind cooperatively to the DNA. Here we have investigated this cooperativity by isolating mutants of the CRP protein that abolish CytR regulation without exhibiting a concomitant loss in their ability to activate transcription. Four different, single amino acid substitutions in CRP give rise to this phenotype. These amino acids lie in close proximity on the surface of the CRP tertiary structure in a portion of the protein that is not in contact with the DNA. In vitro analyses of one of the CRP mutants show that it interacts with the DNA in a manner indistinguishable from wild-type CRP, whereas its interaction with CytR is perturbed. These results strongly indicate that cooperative DNA binding of CytR and cAMP-CRP is achieved through protein-protein interactions.

Characterization of the binding of cAMP and cGMP to the CRP*598 mutant of theE. colicAMP receptor protein

Wild type cAMP receptor protein (CRP) activates in vitro lac transcription only in the presence of cAMP. In contrast the mutant CRP*598 (Arg-142 to His, Ala-144 to Thr) can activate lac transcription in the absence of cyclic nucleotide or at concentrations of cAMP below that required by CRP. To further characterize the properties of CRP*598, the binding of cAMP and cGMP to CRP and CRP*598 has been determined. The intrinsic binding constant (K) values obtained for cAMP binding are: CRP, 1.9 x 10(4) M-1; CRP*598, 3.8 x 10(5) M-1. The K values obtained for cGMP binding are: CRP, 2.9 x 10(4) M-1; CRP*598, 2.7 x 10(4) M-1. The results indicate that the affinity of CRP and CRP*598 for cGMP is relatively unchanged while the affinity of CRP*598 for cAMP is approximately twenty times greater than that shown by CRP. Binding of cAMP by CRP and cGMP by CRP or CRP*598 exhibits slight negative cooperativity. The major difference seen is that CRP*598 binds cAMP with strong positive cooperativity. The importance of the unsubstituted N6 position of the adenine moiety is also shown by the similar affinity of both forms of CRP for N6-butyryl cAMP. The cAMP binding properties evinced by CRP*598 suggest that its intrinsically altered conformation may be related to that assumed by CRP in a CRP-DNA or a cAMP-CRP-DNA complex.

Site-directed mutants of the cAMP receptor protein-DNA binding of five mutant proteins

Oligonucleotide-directed mutagenesis was employed to generate mutants of the cAMP receptor protein (CRP) of Escherichia coli. The mutant proteins were purified to homogeneity and tested for stability and DNA binding. It is shown that mutations at the position of Arg180 abolish specific DNA binding, whereas those at the position Arg185 have very little effect. Both positions have previously been implicated as crucial for the specific interaction between CRP and DNA. The Ser128----Ala mutant shows a slight reduction in DNA binding affinity relative to wild-type. All mutants investigated show similar stability profiles to wild-type CRP with respect to thermolysin proteolysis as a function of temperature.

Structure-function analysis of three cAMP-independent forms of the cAMP receptor protein.

cAMP receptor protein (CRP)-dependent operon expression in Escherichia coli requires the CRP X cAMP complex form of wild-type CRP. One class of crp mutants (crp*) activates CRP-dependent promoters in strains (cya) incapable of endogenous cAMP synthesis. Of fundamental interest is the difference in regulatory properties exhibited by crp* mutant strains, some of which exhibit glucose-mediated repression of beta-galactosidase synthesis, some of which do not. To gain a better understanding of the mechanisms of cAMP-independent promoter activation and repression we have: determined through cloning and DNA sequence analysis the primary structure of three CRP* forms of CRP; purified the mutant proteins; characterized the effect of these mutations on CRP secondary structure; and studied CRP*-activated lac promoter regulation in a purified in vitro transcription system. The results of this study provide strong evidence that mutations in crp alter the conformation of CRP and result in cAMP-independent activation of CRP-dependent promoters in vitro. In addition, a CRP allele-specific inhibition of CRP* activity by spermidine was observed in vitro that parallels crp* strain-specific sensitivity to glucose-mediated repression of CRP-dependent enzyme synthesis in vivo. This observation provides evidence that catabolite repression in cells lacking cAMP may be mediated through a mechanism that inhibits CRP* activity.

Mechanism of CRP-mediated cya suppression in Escherichia coli.

Escherichia coli strain NCR30 contains a cya lesion and a second-site cya suppressor mutation that lies in the crp gene. NCR30 shows a pleiotropic phenotypic reversion to the wild-type state in expressing many operons that require the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex for positive control. In vivo beta-galactosidase synthesis in NCR30 was sensitive to glucose-mediated repression, which was relieved not only by cAMP but also by cyclic GMP and cyclic CMP. The CRP isolated from NCR30 differed from the protein isolated from wild-type E. coli in many respects. The mutant protein bound cAMP with four to five times greater affinity than wild-type CRP. Protease digestion studies indicated that native NCR30 CRP exists in the cAMP-CRP complex-like conformation. The protein conferred a degree of cAMP independence on the in vitro synthesis of beta-galactosidase. In addition, the inherent positive control activity of the mutant protein in vitro was enhanced by those nucleotides that stimulate in vivo beta-galactosidase synthesis in NCR30. The results of this study supported the conclusion that the crp allele of NCR30 codes for a protein having altered effector specificity yet capable of promoting positive control over catabolite-sensitive operons in the absence of an effector molecule.

Phosphoenolpyruvate:sugar phosphotransferase system-mediated regulation of carbohydrate metabolism in Salmonella typhimurium.

The crr mutation was shown to affect the phosphoenolpyruvate:sugar phosphotransferase system-mediated transient repression of the lac operon, intracellular cAMP levels, and sensitivity to inducer exclusion. Our results indicate that the presumed crr gene product, factor IIIGlc, plays a direct role in the regulation of inducer exclusion. We propose a mechanism in which inducer exclusion depends on both the level and state of phosphorylation of factor IIIGlc and the level of an inducer exclusion-sensitive transport system. The results of studies on the sensitivity to inducer exclusion of glycerol and maltose in cultures induced for short periods of time on these substrates (resulting in varying degrees of activity of the respective transport systems) support this model of inducer exclusion. Previously, the crp*-771 mutation has been shown to result in an altered cAMP receptor protein, which has a changed affinity for cAMP, and to affect the sensitivity for inducer exclusion of glycerol. Changes in other functions of the altered cAMP receptor protein were indicated by our results; these changes were in the roles of this protein in (i) the cAMP-dependent initiation of transcription of the lac operon and (ii) the regulation of intracellular cAMP levels and the export of cAMP. We propose that the crp*-771 mutation has an indirect effect in relieving inducer exclusion in repressed or hypersensitive strains, in which the crp*-771 mutation allows the synthesis of inducer exclusion-sensitive transport systems to higher levels than the levels found in strains containing wild-type cAMP receptor protein.