Kinetic Studies of cAMP-induced Allosteric Changes in Cyclic AMP Receptor Protein from Escherichia coli

Małecki, Jędrzej; Polit, Agnieszka; Wasylewski, Zygmunt

doi:10.1074/jbc.275.12.8480

Cited by 33 publications

(58 citation statements)

References 26 publications

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“…Biochemical and biophysical studies focused on the identification of structural changes accompanying binding of cAMP to CRP have been mostly performed under equilibrium conditions, and much less attention has been paid to the description of the kinetic mechanism of the ligand interaction with CRP. Our previous stopped-flow studies investigating the kinetics of conformational changes in CRP induced by cAMP binding had shown that, at micromolar cAMP concentrations, allosteric changes take place according to a sequential (Koshland-Ném-ethy-Filmer (KNF)) model, whereas conformational changes observed at millimolar concentrations can be described by a concerted (Monod-Wyman-Changeux (MWC)) model (8). The conformational change observed upon binding of cAMP to antisites, described by the KNF model, clearly indicates long-range structural communication between the N-and C-terminal domains of CRP remaining under kinetic control (8).…”

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“…Our previous stopped-flow studies investigating the kinetics of conformational changes in CRP induced by cAMP binding had shown that, at micromolar cAMP concentrations, allosteric changes take place according to a sequential (Koshland-Ném-ethy-Filmer (KNF)) model, whereas conformational changes observed at millimolar concentrations can be described by a concerted (Monod-Wyman-Changeux (MWC)) model (8). The conformational change observed upon binding of cAMP to antisites, described by the KNF model, clearly indicates long-range structural communication between the N-and C-terminal domains of CRP remaining under kinetic control (8). On the other hand, binding of cAMP to syn-sites of CRP, described by the MWC model, results from displacement of equilibrium between the two structural forms of CRP⅐cAMP 2 , where only one form is able to form a CRP⅐cAMP 4 complex.…”

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“…On the other hand, binding of cAMP to syn-sites of CRP, described by the MWC model, results from displacement of equilibrium between the two structural forms of CRP⅐cAMP 2 , where only one form is able to form a CRP⅐cAMP 4 complex. Because the four cAMPbinding sites of CRP possess different cooperative affinity for the ligand (8), it can be expected that the kinetically controlled domain-domain and subunit-subunit communication may play a crucial role in the molecular regulatory mechanism of CRP action. In this study, we report a further kinetic description of cAMP-induced conformational changes in CRP based on the use of different mutants (T127I, S128A, and T127I/S128A).…”

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“…Indeed, a recent microcalorimetric isothermal titration calorimetry study has shown that, in solution, CRP sequentially binds two molecules of cAMP with negative cooperativity and high affinity; and additionally, at least one additional molecule binds to a low affinity binding site (5). It is believed that, at ϳ100 M cAMP, CRP is able to recognize and bind specific DNA sequences and to stimulate transcription (6), and this active form of the protein is represented by the CRP⅐cAMP 2 complex with two anti-sites occupied (7,8). However, in the presence of millimolar concentrations of cAMP, at which the CRP⅐cAMP 4 complex predominates, there is a loss of affinity for DNA and sequence specificity for its recognition and transcription regulation (7).…”

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

Polit

Bonarek

Kepys

et al. 2003

Journal of Biological Chemistry

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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...

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

Polit

Bonarek

Kepys

et al. 2003

Journal of Biological Chemistry

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“…It was reported that Trp-13 is substantially solventexposed and contributes to ϳ80% of the Trp fluorescence, whereas Trp-85 is buried in the matrix of CRP and contributes to roughly 20% of the Trp fluorescence (44). Results obtained using single-tryptophan-containing CRP mutants indicate that Trp-85 is accountable for the total change observed of the intrinsic Trp fluorescence in wild-type CRP (45). The abovecited reports all strongly suggest that the significant changes on the tryptophan fluorescence in the deletion and the double mutants are most likely attributable to Trp-85.…”

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confidence: 56%

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

Chen

Lee

2003

Journal of Biological Chemistry

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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 qua- Cyclic AMP serves as an intracellular message in both prokaryotes and eukaryotes by transmitting information through proteins such as protein kinase A (PKA) 1 , cyclic nucleotidegated ion channels (CNGC), and cAMP receptor protein in Escherichia coli (CRP). These proteins are involved in a very diverse set of cellular functions such as signal transduction, excitability, and gene expression (1-6). These proteins of diverse functions all consist of a cAMP binding motif. The structural motif, which serves as cAMP receptor, is found to display a high degree of similarity. X-ray crystallography and homology modeling results show that, despite obvious divergence of sequence among the receptor domains and significantly different biological functions of these proteins, their CNB domains appear to share a common architecture, all consisting of an ␣-helix (helix A), an eight-stranded ␤-roll, and two more ␣-helices (helices B and C). The body of the CNB pocket is mainly located in the ␤-roll, with the C-helix forming the back of the binding pocket (2,8). The superimposition of the structures of CNB domains from CRP and the regulatory subunits of PKA, as shown below in Fig. 1, indicates that the ␤-roll basically assumes the same structure with the exception of loops 3 and 4 between strands 4 and 5, and strands 6 and 7, respectively. In some cases, such as in CNGC and PKA, loop 3 is shortened whereas loop 4 is lengthened, as shown in Fig. 1. Only six residues (Gly-33, Gly-45, Gly-71, Glu-72, Arg-82, and Ala-84 using the CRP sequence as reference) are invariant among all members of the families. It has been suggested that the invariant residues play important and conserved roles in the folding and function of the CNB sites of these diverse proteins. Gly-33, Gly-45, and Gly-71 are involved in turns between strands of the ␤-roll; Arg-82 and Glu-72 contact the cyclic nucleotide, and the function of Ala-84 is uncertain (3). Despite large variation of primary sequences, the sizes of secondary structural elements of the CNB domain are much conserved among the family. For example, the alignment of CRP and CNGCs by keeping the six conserve residues at the same positions shows that the si...

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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

Fujimoto¹,

Toyama²,

Takeuchi³

2002

Biopolymers

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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.

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Kinetic Studies of cAMP-induced Allosteric Changes in Cyclic AMP Receptor Protein from Escherichia coli

Cited by 33 publications

References 26 publications

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

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

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

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

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