Proton nuclear magnetic resonance studies on cyclic nucleotide binding to the Escherichia coli adenosine cyclic 3',5'-phosphate receptor protein

Gronenborn, Angela M.; Clore, G. Marius

doi:10.1021/bi00260a020

Cited by 66 publications

(28 citation statements)

References 38 publications

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“…The good agreement between this conformation of unligated CRP and the experimental SANS curve does imply that this energy-minimized open form may indeed be the stable conformation of unligated CRP in solution. These results agree with NMR measurements, which show a tightening up of CRP into a more rigid protein upon cAMP binding (2). The magnitude of the change of approximately 1 Å in the radius of gyration also agrees with the 0.7 Ϯ 0.1 Å shrinkage observed in the Stokes radius of CRP upon ligation with cAMP (22).…”

supporting

confidence: 89%

“…The implication is that the asymmetry of the CRP dimer in the crystal lattice is due to crystal packing forces and that the structural change responsible for the activation of transcription is a change from the open to the closed form in solution. This is substantiated by NMR measurements on fluorinated derivatives of CRP, which imply that the conformational change involves the hinge region (1), and proton NMR measurements, which show that CRP in solution tightens up and becomes more rigid upon binding of cAMP (2).…”

mentioning

confidence: 79%

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Determination of the Conformations of cAMP Receptor Protein and Its T127L,S128A Mutant with and without cAMP from Small Angle Neutron Scattering Measurements

Krueger¹,

Gorshkova

Brown

et al. 1998

Journal of Biological Chemistry

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The binding of 3Ј,5Ј cyclic adenosine monophosphate (cAMP) to cAMP receptor protein (CRP) 1 activates the transcription of over 20 genes that code for the catabolite enzymes involved in carbohydrate metabolism in Escherichia coli. Results from NMR (1, 2), Raman spectroscopy (3), proteolysis studies (4), small angle x-ray scattering measurements (5), and isothermal titration calorimetry (6) indicate that upon binding to cAMP, CRP undergoes a conformational change to a conformation that promotes specific binding to the catabolite operons to activate transcription in the presence of RNA polymerase. The nature of this conformational change is not known, because only the structures of the cAMP-ligated CRP complex and of the DNAcAMP-ligated CRP complex have been determined from x-ray crystallography studies (7,8).CRP exists as a homodimer of 45,000 g mol Ϫ1 consisting of a ␤-pleated sheet amino-terminal domain, which contains the cAMP binding site and a carboxyl-terminal domain consisting of several ␣-helices that bind to specific DNA sequences. In the crystal phase, the cAMP-ligated CRP dimer is asymmetric where one monomer is in an "open" form in which the ␣-helices are swung out away from the amino-terminal domain and the other monomer is in a "closed" form in which the ␣-helices are swung in close to the amino-terminal domain (7). The amino acid sequence connecting the two domains is, thus, called the "hinge" region. The x-ray crystal structure of the cAMP-ligated CRP dimer complexed with a 30-base pair DNA sequence is exclusively in the closed form (8). Recent energy minimization computations of the two forms in solution show that the lowest energy conformation of the cAMP-ligated CRP dimer is exclusively the closed form in solution (9). The implication is that the asymmetry of the CRP dimer in the crystal lattice is due to crystal packing forces and that the structural change responsible for the activation of transcription is a change from the open to the closed form in solution. This is substantiated by NMR measurements on fluorinated derivatives of CRP, which imply that the conformational change involves the hinge region (1), and proton NMR measurements, which show that CRP in solution tightens up and becomes more rigid upon binding of cAMP (2).There is, however, evidence that the conformational change may not simply involve a transition from the open to the closed form of CRP upon binding to cAMP in solution. A comparison of the secondary structure of free CRP and cAMP-ligated CRP by Raman spectroscopy (3) shows a structural shift from 44% ␣-helix, 28% ␤-strand, 18% turn, and 10% undefined in the free form to 37% ␣-helix, 33% ␤-strand, 17% turn, and 12% undefined in the ligated form, implying that the conformational change does not conserve the ␣-helical and ␤-strand structure as it would in a shift of the CRP dimer from the exclusively open to the closed form in solution. A conformational change involving alterations in the ␣-helical and ␤-strand structure of CRP is also implied by an empirical model of CR...

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supporting

confidence: 89%

mentioning

confidence: 79%

Determination of the Conformations of cAMP Receptor Protein and Its T127L,S128A Mutant with and without cAMP from Small Angle Neutron Scattering Measurements

Krueger¹,

Gorshkova

Brown

et al. 1998

Journal of Biological Chemistry

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“…3 also appears to rule out the hypothesis (32) that CAP-bound cAMP interacts directly with a DNA base pair in the recognition site. Although the results of crystallographic (21) and solution (33,34) experiments conflict regarding the glycosidic conformation of CAP-bound cAMP, both types of experiment agree that the cAMP binding site is located in a ,/roll structure comprised of amino acids 18-97 of CAP (21,34,35). In the alignment illustrated, the cAMP binding site is seen to be a minimum of 25 A from the DNA, thereby precluding a cAMP-DNA contact.…”

Section: And 6)mentioning

confidence: 96%

Molecular basis of DNA sequence recognition by the catabolite gene activator protein: detailed inferences from three mutations that alter DNA sequence specificity.

Ebright¹,

Cossart²,

Gicquel-Sanzey³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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Previously, we reported that substitution of Glu-181 of the catabolite gene activator protein (CAP) by lysine, leucine, or valine results in a protein that has specificity for APT base pairs at positions 7 and 16 of the DNA recognition site, rather than GC base pairs as is the case with the wild-type CAP. In this paper, we deduce from these genetic data both (i) the specific chemical interactions by which amino acid side chains at position 181 interact with base pairs 7 and 16 and (ii) the precise alignment between the structures of the CAP and DNA in the intermolecular CAP-DNA complex. Our analysis supports the idea that the two symmetry-related The three-dimensional structures of three proteins that bind to specific DNA sequences have been determined recentlyi.e., the catabolite gene activator protein (CAP) complex with cAMP (1), cro (2), and the amino-terminal fragment of the X repressor (ref. 3; reviewed in ref. 4). Because the structures determined were those of the uncomplexed proteins, efforts to date to elucidate the structures of the protein-DNA complexes have relied on model building (1)(2)(3)(4)(5)(6)(7)(8). The models proposed in the cases of cro and the X repressor and one of four models in the case of CAP exhibit common features. Each model postulates that a pair of 2-fold related ahelices, one from each subunit of the protein, interacts with successive major grooves of right-handed B-type DNA (2, 3, 7). As discussed below, in each instance the a-helix proposed to contact DNA is the second a-helix of a characteristic helix-turn-helix structural motif (9-13). In this paper, we discuss the implications for this analysis of our genetic results (14), which we believe identify a direct contact between an amino acid of a DNA binding protein and the base pair it contacts in the target DNA sequence.

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“…Thus, the crystal structures reveal cAMP binds in an anti conformation to CAP (3) but in a syn conformation to PKA RI␣ (4), although this may not reflect the conformation of the ligand bound to the proteins in solution (1,2,19). While experiments with cyclic nucleotide analogs and modeling, based upon the CAP and PKA R1␣ structures, have been used to investigate the conformation adopted by agonists in other CNB sites, this issue is unresolved (1)(2)(3)(4)(5)(6).…”

mentioning

confidence: 99%

A State-independent Interaction between Ligand and a Conserved Arginine Residue in Cyclic Nucleotide-gated Channels Reveals a Functional Polarity of the Cyclic Nucleotide Binding Site

Tibbs

Liu

Leypold

et al. 1998

Journal of Biological Chemistry

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Activation of cyclic nucleotide-gated channels is thought to involve two distinct steps: a recognition event in which a ligand binds to the channel and a conformational change that both opens the channel and increases the affinity of the channel for an agonist. Sequence similarity with the cyclic nucleotide-binding sites of cAMP-and cGMP-dependent protein kinases and the bacterial catabolite activating protein (CAP) suggests that the channel ligand binding site consists of a ␤-roll and three ␣-helices. Recent evidence has demonstrated that the third (or C) ␣-helix moves relative to the agonist upon channel activation, forming additional favorable contacts with the purine ring. Here we ask if channel activation also involves structural changes in the ␤-roll by investigating the contribution of a conserved arginine residue that, in CAP and the kinases, forms an important ionic interaction with the cyclized phosphate of the bound ligand. Mutations that conserve, neutralize, or reverse the charge on this arginine decreased the apparent affinity for ligand over four orders of magnitude but had little effect on the ability of bound ligand to open the channel. These data indicate that the cyclized phosphate of the nucleotide approaches to within 2-4 Å of the arginine, forming a favorable ionic bond that is largely unaltered upon activation. Thus, the binding site appears to be polarized into two distinct structural and functional domains: the ␤-roll stabilizes the ligand in a state-independent manner, whereas the C-helix selectively stabilizes the ligand in the open state of the channel. It is likely that these distinct contributions of the nucleotide/C-helix and nucleotide/␤-roll interactions may also be a general feature of the mechanism of activation of other cyclic nucleotide-binding proteins.Cyclic nucleotides regulate the activity of a diverse family of proteins involved in cellular signaling. These include a transcription factor (the bacterial catabolite activating protein, CAP), the cAMP-(PKA) 1 and cGMP-dependent protein kinases (PKG) and the cyclic nucleotide-gated (CNG) ion channels involved in visual and olfactory signal transduction (1, 2). Despite obvious divergence among the effector domains of these proteins, the cyclic nucleotide binding (CNB) sites appear to share a common architecture. Solution of the crystal structures of CAP (3) and a recombinant bovine PKA RI␣ subunit (4) has demonstrated that their CNB sites are formed from an ␣-helix (A helix), an 8-stranded ␤-roll, and two more ␣-helices (B and C), with the C-helix forming the back of the binding pocket. Six residues are invariant among all members of the CAP and kinase families: three glycines involved in turns between strands of the ␤-roll, an arginine and a glutamate, each of which contact the cyclic nucleotide, and an alanine whose function is uncertain (1) (see also Fig. 1). Strikingly, these six residues are conserved in the CNG channels. Thus, it has been suggested that the invariant residues play important and conserved roles in the fol...

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Proton nuclear magnetic resonance studies on cyclic nucleotide binding to the Escherichia coli adenosine cyclic 3',5'-phosphate receptor protein

Cited by 66 publications

References 38 publications

Determination of the Conformations of cAMP Receptor Protein and Its T127L,S128A Mutant with and without cAMP from Small Angle Neutron Scattering Measurements

Determination of the Conformations of cAMP Receptor Protein and Its T127L,S128A Mutant with and without cAMP from Small Angle Neutron Scattering Measurements

Molecular basis of DNA sequence recognition by the catabolite gene activator protein: detailed inferences from three mutations that alter DNA sequence specificity.

A State-independent Interaction between Ligand and a Conserved Arginine Residue in Cyclic Nucleotide-gated Channels Reveals a Functional Polarity of the Cyclic Nucleotide Binding Site

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