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At least 19 recordsLinked to original sources
PubMed2002

Stoichiometry and structural effect of the cyclic nucleotide binding to cyclic AMP receptor protein.

Cyclic AMP receptor protein (CRP) is a homodimeric protein, which is activated by cAMP binding to function as a transcriptional regulator of many genes in prokaryotes. Until now, the actual number of cAMP molecules that can be bound by CRP in solution has been ambiguous. In this work, we performed a nuclear magnetic resonance study on CRP to investigate the stoichiometry of cyclic nucleotide binding to CRP. A series of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of the protein in the absence and in the presence of cAMP or cGMP were analyzed. The addition of cAMP to CRP induced a biphasic spectral change up to 4 equivalents, whereas the cGMP addition made a monophasic change up to 2 equivalents. Altogether, the results not only established for the first time that CRP possesses two cyclic AMP-binding sites in each monomer, even in a solution without DNA, but also suggest that the syn-cAMP binding sites of the CRP dimer can be formed by an allosteric conformational change of the protein upon the binding of two anti-cAMPs at the N-terminal domain. In addition, a residue-specific inspection of the spectral changes provides some new structural information about the cAMP-induced allosteric activation of CRP.

Allosteric Regulation↗
PubMed2002

Ligand-induced conformational and structural dynamics changes in Escherichia coli cyclic AMP receptor protein.

Cyclic AMP receptor protein (CRP) regulates the expression of a large number of genes in E. coli. It is activated by cAMP binding, which leads to some yet undefined conformational changes. These changes do not involve significant redistribution of secondary structures. A potential mechanism of activation is a ligand-induced change in structural dynamics. Hence, the cAMP-mediated conformational and structural dynamics changes in the wild-type CRP were investigated using hydrogen-deuterium exchange and Fourier transform infrared spectroscopy. Upon cAMP binding, the two functional domains within the wild-type CRP undergo conformational and structural dynamics changes in two opposite directions. While the smaller DNA-binding domain becomes more flexible, the larger cAMP-binding domain shifts to a less dynamic conformation, evidenced by a faster and a slower amide H-D exchange, respectively. To a lesser extent, binding of cGMP, a nonfunctional analogue of cAMP, also stabilizes the cAMP-binding domain, but it fails to mimic the relaxation effect of cAMP on the DNA-binding domain. Despite changes in the conformation and structural dynamics, cAMP binding does not alter significantly the secondary structural composition of the wild-type CRP. The apparent difference between functional and nonfunctional analogues of cAMP is the ability of cAMP to effect an increase in the dynamic motions of the DNA binding domain.

Binding Sites↗
PubMed2000

Structural understanding of the allosteric conformational change of cyclic AMP receptor protein by cyclic AMP binding.

Cyclic AMP receptor protein (CRP) plays a key role in the regulation of more than 150 genes. CRP is allosterically activated by cyclic AMP and binds to specific DNA sites. A structural understanding of this allosteric conformational change, which is essential for its function, is still lacking because the structure of apo-CRP has not been solved. Therefore, we performed various NMR experiments to obtain apo-CRP structural data. The secondary structure of apo-CRP was determined by analyses of the NOE connectivities, the amide proton exchange rates, and the (1)H-(15)N steady-state NOE values. A combination of the CSI-method and TALOS prediction was also used to supplement the determination of the secondary structure of apo-CRP. This secondary structure of apo-CRP was compared with the known structure of cyclic AMP-bound CRP. The results suggest that the allosteric conformational change of CRP caused by cyclic AMP binding involves subunit realignment and domain rearrangement, resulting in the exposure of helix F onto the surface of the protein. Additionally, the results of the one-dimensional [(13)C]carbonyl NMR experiments show that the conformational change of CRP caused by the binding of cyclic GMP, an analogue of cyclic AMP, is different from that caused by cyclic AMP binding.

Allosteric Regulation↗
PubMed2002

Binding modes of cyclic AMP and environments of tryptophan residues in 1:1 and 1:2 complexes of cyclic AMP receptor protein and cyclic AMP.

Cyclic AMP (cAMP) receptor protein (CRP) forms 1:1 and 1:2 complexes with cAMP, and the former complex is considered to be the most active form of CRP in binding to specific DNA sequences and in modulating gene transcription by RNA polymerases. We examine the cAMP binding modes and structural changes of CRP upon cAMP binding by UV resonance Raman spectroscopy. The Raman spectra of CRP-(cAMP)(1) and CRP-(cAMP)(2) extracted from those of CRP-cAMP mixtures at varied mixing ratios clearly show that the hydrogen bonding state and the conformation of cAMP in both complexes in solution are very similar to those found in the X-ray crystal structure of CRP-(cAMP)(2), which is evidence that the cAMP binding mode does not differ between the two complexes. The environmental hydrophobicity of Trp85 monitored by UV resonance Raman intensity shows a significant decrease upon binding of the first cAMP molecule, whereas no further change occurs in the second cAMP binding step. The environmental change of Trp85 suggests an opening of the cleft between the N-terminal cAMP and C-terminal DNA binding domains in the process of CRP activation by binding of a single cAMP molecule.

Cyclic AMP↗
PubMed2003

Kinetics of transcription initiation at lacP1. Multiple roles of cyclic AMP receptor protein.

The cyclic AMP receptor protein (CRP) acts as a transcription activator at many promoters of Escherichia coli. We have examined the kinetics of open complex formation at the lacP1 promoter using tryptophan fluorescence of RNA polymerase and DNA fragments with 2-aminopurine substituted at specific positions. Apart from the closed complex formation and promoter clearance, we were able to detect three steps. The first step after the closed complex formation leads to a rapid increase of 2-aminopurine fluorescence. This was followed by another rapid step in which quenching of tryptophan fluorescence of RNA polymerase was observed. The slowest step detected by 2-aminopurine fluorescence increase is assigned to the final open complex formation. We have found that CRP not only enhances RNA polymerase binding at the promoter, but also enhances the slowest isomerization step by about 2-fold. Furthermore, potassium permanganate probing shows that the conformation of the open complex in the presence of CRP appears qualitatively and quantitatively different from that in the absence of CRP, suggesting that contact with RNA polymerase is maintained throughout the transcription initiation.

2-Aminopurine↗
PubMed1983

Cyclic AMP receptor protein and cyclic AMP-dependent protein kinase activity in rabbit peritoneal neutrophils.

The cAMP receptor protein and cAMP-dependent protein kinase activity in rabbit peritoneal neutrophils have been identified. The cAMP receptor protein in either the plasma membrane or cytosol fractions, identified by photoaffinity labeling with 8-N3-[32P]cAMP, has an apparent molecular weight of 54,000. The cytosol and membrane receptor proteins have apparent dissociation constants for 8-N3-[32P]cAMP of 0.20 microM and 0.06 microM, respectively. The molecular weight and dissociation constant for 8-N3-[32P]cAMP of this cAMP receptor protein are similar to what has been known for RII, the regulatory subunit of the type II cAMP-dependent protein kinase. Unlike the human neutrophils, no evidence of RI activity was detected. cAMP-dependent protein kinase activity was identified by using histone as a substrate. Subcellular fractionation studies showed that the cAMP receptor protein and the cAMP-dependent protein kinase activity are most enriched in the cytosol fraction.

Animals↗
PubMed1981

Species specificity of brain cyclic AMP receptor proteins.

The cyclic AMP binding proteins present in mouse, rat, bovine, and sheep brains were compared. Extracts were isotopically labeled with 8-azido-cyclic [32P]AMP, a photoaffinity analog specific for cyclic AMP binding sites, and then subjected to two-dimensional gel electrophoresis. The resulting autoradiographic patterns were generally similar, but showed consistent species variations. Proteins identified by their size and phosphorylatability as regulatory subunits of Type II protein kinase isozymes were present in all species, but with slight variations in pI. A series of charge variants identified as regulatory subunits of Type I kinase isozymes on the basis of their size was also ubiquitously present, as were several smaller proteins postulated to be proteolytic fragments derived from the regulatory subunits. The major species difference was a series of labeled proteins found only in rodent brains and not in the brains of any ruminant, or in other rodent tissues. These proteins had a molecular weight of 54,000 and a pI range of 5.89--6.26, and could not be endogenously phosphorylated. The identities of these proteins and their relationship to the protein kinase regulatory subunits are unknown.

Animals↗
PubMed2000

Kinetic studies of cAMP-induced allosteric changes in cyclic AMP receptor protein from Escherichia coli.

Cyclic AMP receptor protein (CRP) regulates the expression of several genes in Escherichia coli. The ability of CRP to bind specific DNA sequences and stimulate transcription is achieved as result of binding of an allosteric ligand: cAMP. Stopped-flow fluorimetry was employed to study the kinetics of the conformational changes in CRP induced by cAMP binding to high and low affinity receptor sites. Results of experiments using CRP labeled at Cys-178 with 1,5-I-AENS indicate change in conformation of the helix-turn-helix, occurring after the formation of CRP-cAMP(2) complex, i.e. after saturation of the high affinity sites. The observed conformational change occurs according to sequential model of allostery and is described by rate constants: k(c) = 9.7 +/- 0.1 s(-1) and k(-c) = 0.31 +/- 0.05 s(-1), for the forward and backward reaction, respectively. Results of experiments monitored using CRP intrinsic fluorescence suggest that conformational change precedes the formation of CRP-cAMP(4) complex and results from displacement of equilibrium between two forms of CRP-cAMP(2), caused by binding of cAMP to low affinity sites of one of these forms only. The observed conformational change occurs according to concerted model of allostery and is described by rate constants: k(on) = 28 +/- 1.5 s(-1) and k(off) = 75.5 +/- 3 s(-1). Results of experiments using single-tryptophan-containing CRP mutants indicate that Trp-85 is mainly responsible for the observed total change in intrinsic fluorescence of wild-type CRP.

Allosteric Regulation↗
PubMed1999

Functional roles of the two cyclic AMP-dependent forms of cyclic AMP receptor protein from Escherichia coli.

The cyclic AMP receptor protein activates transcription in Escherichia coli, only when complexed with cyclic AMP. The cyclic AMP receptor protein-cyclic AMP complex formed at low concentrations of cyclic AMP has a different conformation from either cyclic AMP receptor protein alone or its complex with cyclic AMP formed at high cyclic AMP concentrations. Various biophysical data suggest that the latter complex resembles free cyclic AMP receptor protein. We have examined the conformational and biological properties of cyclic AMP receptor protein as a function of cyclic AMP concentrations, using the gal operon of E. coli. A biphasic behavior is observed. It is shown that only the complex formed at lower concentrations of cyclic AMP is the transcriptionally active form. This difference between the complexes at different levels of cyclic AMP arises from a decreased ability of the cyclic AMP receptor protein-cyclic AMP complex at high cyclic AMP concentrations to bind to DNA at specific sites.

Bacterial Proteins↗
PubMed2002

Structure and dynamics of the modular halves of Escherichia coli cyclic AMP receptor protein.

E. coli cyclic AMP receptor protein, CRP, is a modular protein that consists of a covalent linkage of two common structural domains. To probe the mechanism for intramolecular communications and to define the unique properties acquired by covalent linkage, the structural, and functional properties of the cAMP- and DNA-binding domains of CRP were studied separately as two independent polypeptides. The N-terminal cAMP-binding domain (alpha-CRP), including S-CRP and CH-CRP, which were generated by digestion of CRP by subtilisin and chymotrypsin, respectively, are mainly populated by beta-sheets. The C-terminal DNA-binding domain, designated as beta-CRP, consists of mostly alpha-helices. The residues of S-CRP and CH-CRP are from 1 to 116 and 1 to 136 of intact wild-type CRP, and those of beta-CRP are from 108 to 209. The secondary structures of alpha-CRP and beta-CRP were monitored by FT-IR, and they are similar to those of the corresponding parts in intact wild-type CRP. Results from hydrogen-deuterium exchange experiments indicated that beta-CRP is more dynamic than alpha-CRP. In an earlier study, it was shown that alpha-CRP retains the function of binding cAMP [Heyduk, E., et al. (1992) Biochemistry 31, 3682-3688]. beta-CRP was able to bind to DNA, although only weakly, and was not sequence specific. Thus, a covalent linkage between the two domains is essential for the realization of the intramolecular signal transmission between the domains triggered by ligand binding. The acquisition of this unique property is intimately associated with the dynamics of the molecule.

Amino Acid Motifs↗
PubMed2002

Ability of E. coli cyclic AMP receptor protein to differentiate cyclic nucelotides: effects of single site mutations.

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.

Cyclic AMP↗
PubMed2001

Detection of the protein-protein interaction between cyclic AMP receptor protein and RNA polymerase, by (13)C-carbonyl NMR.

Cyclic AMP receptor protein (CRP) plays a key role in the transcription regulation of many prokaryotic genes. Upon the binding of cyclic AMP, CRP is allosterically activated, binds to target DNA sites, and interacts with RNA polymerase. Although the protein-protein interaction between CRP and RNA polymerase is known to be important for the transcription initiation of the target genes, its structural understanding is still lacking, particularly due to the high molecular mass (approximately 120 kDa) of the protein complex. We assigned all of the (13)C-carbonyl resonances of methionine residues in CRP by using the double labeling and the enzyme digestion techniques. The result of (13)C-carbonyl NMR experiment on [(13)C'-Met]-CRP in the presence of both cyclic AMP and RNA polymerase alpha subunit showed that the two proteins interact with each other in solution in the absence of DNA via the region around the residues from Met 157 to Met 163 in CRP. The results also showed the effectiveness of the selective labeling and (13)C-carbonyl NMR spectroscopy in the specific detection of the protein-protein interaction between large molecules.

Allosteric Regulation↗
PubMed1988

Cyclic AMP-induced conformational change of cyclic AMP receptor protein (CRP): intragenic suppressors of cyclic AMP-independent CRP mutations.

We isolated and characterized crp mutations in Escherichia coli that allow cyclic AMP (cAMP) receptor protein to function without cAMP. These mutants defined a region involved in the cAMP-induced allosteric change of cAMP receptor protein that is necessary for activation of the protein. Currently, we have isolated intragenic suppressors of the crp mutations. These crp (Sup) mutants require cAMP for activity. The crp (Sup) mutations map in regions which define new sites of changes involved in cAMP receptor protein activation. From these results, we suggest that to activate cAMP receptor protein cAMP brings about (i) a hinge reorientation to eject the DNA-binding F alpha-helices, (ii) proper alignment between the two subunits, and (iii) an adjustment between the position of the two domains. Cyclic GMP fails to effect the last step.

Allosteric Site↗
PubMed1996

Protein-protein interactions during transcription activation: the case of the Escherichia coli cyclic AMP receptor protein.

The Escherichia coli cyclic AMP receptor protein (CRP) is a homodimeric transcription activator triggered by cyclic AMP. Escherichia coli contains more than 100 different promoters that can be activated by CRP: in most cases the CRP acts by making direct contact with RNA polymerase. Remarkably, there is considerable variation in the location of the DNA site for CRP from one CRP-dependent promoter to another. Genetic methods have been used to locate the activating regions of CRP that make contact with RNA polymerase at promoters of different architectures. At promoters where the DNA site for CRP is centred near to positions -61, -71 or -81 (i.e. 61, 71 or 81 base pairs upstream of the transcript start-point, respectively), a single surface-exposed loop (Activating Region 1) in the downstream subunit of the CRP dimer makes contact with RNA polymerase. The contact site in RNA polymerase is located in one of the C-terminal domains of two RNA polymerase alpha subunits. At promoters where the DNA site for CRP is centred near to position-41, both subunits of the CRP dimer make contact with RNA polymerase via three separate surface exposed regions (Activating Regions 1, 2 and 3). At these promoters, where bound CRP overlaps with RNA polymerase-binding elements, the C-terminal domains of the polymerase alpha subunits are displaced and bind upstream of CRP. Activation at a number of E. coli promoters is dependent on binding of two CRP dimers, with one dimer bound near to position-41 and the other dimer bound further upstream. In these cases, both bound CRP dimers contact RNA polymerase. The CRP dimer bound around position-41 contacts RNA polymerase via Activating Regions 1, 2 and 3, whereas the upstream bound CRP dimer contacts one of the displaced alpha C-terminal domains via Activating Region 1 in the downstream CRP subunit. Thus in these cases, codependence on two activators is due to simultaneous contacts between separate activators and RNA polymerase. This mechanism allows great flexibility, as any activator that can contact the C-terminal domain of the RNA polymerase alpha subunits can act cooperatively with CRP.

Binding Sites↗
PubMed2004

Measurement of DNA helical change for the binding of cyclic AMP receptor protein to lac DNA.

Cyclic AMP receptor protein (CRP) binds to DNA and induces DNA bending. DNA bending plays an important role in transcription activation of many genes in Escherichia coli. To know DNA helical change by CRP, we used molar cyclization factor (j(M)), which is sensitive to helical twist or torsional alignment. Six consecutive DNA fragments were constructed that contained phasing sequence at left arm and CRP binding site in center. j(M) values measured in this study were approximately 10 times increased by CRP relative to that measured for DNA only. The analysis of set of j(M) values indicated that CRP induced DNA bending without the helical change like helical twisting or torsional alignment change.

Base Sequence↗
PubMed2003

Functional roles of loops 3 and 4 in the cyclic nucleotide binding domain of cyclic AMP receptor protein from Escherichia coli.

Cyclic AMP is a ubiquitous secondary message that regulates a large variety of functions. The protein structural motif that binds cAMP is highly conserved with the exception of loops 3 and 4, whose structure and length are variable. The cAMP receptor protein of Escherichia coli, CRP, was employed as a model system to elucidate the functional roles of these loops. Based on the sequence differences between CRP and cyclic nucleotide gated channel, three mutants of CRP were constructed: deletion (residues 54-56 in loop 3 were deleted), insertion (loop 4 was lengthened by 5 residues between Glu-78 and Gly-79) and double mutants. The effects of these mutations on the structure and function of CRP were monitored. Results show that the deletion and insertion mutations do not significantly change the secondary structure of CRP, although the tertiary and quaternary structures are perturbed. The functional data indicate that loop 3 modulates the binding affinities of cAMP and DNA. Although the lengthened loop 4 may have some fine-tuning functions, the specific function of the original loop 4 of CRP remains uncertain. The function consequences of mutation in loop 3 of CRP are similar to that of site A and site B in the regulatory subunits of cyclic AMP-dependent protein kinases. Thus, the roles played by loop 3 in CRP may represent a more common mechanism employed by cyclic nucleotide binding domain in modulating ligand binding affinity and intramolecular communication.

Amino Acid Sequence↗
PubMed1990

A nuclear magnetic resonance study of the cyclic AMP receptor protein (CRP): assignments of the NH protons of histidine and tryptophan residues and the effect of binding of cAMP to CRP.

Seven well-resolved signals could be observed in the field lower than 9.5 ppm in the 1H-NMR spectrum of the H2O solution of cyclic AMP receptor protein (CRP). The signals of the tryptophan and histidine residues were identified on the basis of the CPMG spin echo spectra, the intra-residue NOE, 15N labeling, deuterium labeling, and the results of pH titration. The assignments of peaks to specific tryptophan and histidine residues are discussed in relation to the amino acid sequence and X-ray crystallographic data, and were confirmed by experiments involving partial subtilisin digestion. The four signals E (11.25 ppm), F (11.15 ppm), G (10.75 ppm), and H (10.65 ppm) were tentatively assigned to the resonances of the histidine residue at position 159, the arginine residue at position 82, and the tryptophan residues at positions 85 and 13, respectively. On the addition of cAMP and cGMP, signals F and G shifted up- and downfield respectively and conformational changes in the structure of CRP could be detected. The conformational transition mostly occurs when one cAMP molecule binds to one of the dimer subunits, but is completed only when both cAMP binding sites are saturated.

Carrier Proteins↗