Thermodynamics of Cyclic Nucleotide Binding to the cAMP Receptor Protein and Its T127L Mutant

Gorshkova, Inna; Moore, Julie L.; McKenney, Keith; Schwarz, Frederick P.

doi:10.1074/jbc.270.37.21679

Cited by 60 publications

(81 citation statements)

References 19 publications

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“…As with wild-type CRP, the sequential binding of two cAMP molecules to CRP SC showed an initial exothermic phase followed by an endothermic phase. This biphasic behavior agrees with previous ITC studies (13,22) and corroborates that the polypeptide linker does not affect the affinities nor the thermodynamic signatures associated with cAMP binding.…”

Section: Quantification Of Camp Binding and Dna Interactions In Crp Scsupporting

confidence: 79%

Asymmetric configurations in a reengineered homodimer reveal multiple subunit communication pathways in protein allostery

Lanfranco

Gárate

Engdahl³

et al. 2017

Journal of Biological Chemistry

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Many allosteric proteins form homo-oligomeric complexes to regulate a biological function. In homo-oligomers, subunits establish communication pathways that are modulated by external stimuli like ligand binding. A challenge for dissecting the communication mechanisms in homo-oligomers is identifying intermediate liganded states, which are typically transiently populated. However, their identities provide the most mechanistic information on how ligand-induced signals propagate from bound to empty subunits. Here, we dissected the directionality and magnitude of subunit communication in a reengineered single-chain version of the homodimeric transcription factor cAMP receptor protein. By combining wild-type and mutant subunits in various asymmetric configurations, we revealed a linear relationship between the magnitude of cooperative effects and the number of mutant subunits. We found that a single mutation is sufficient to change the global allosteric behavior of the dimer even when one subunit was wild type. Dimers harboring two mutations with opposite cooperative effects had different allosteric properties depending on the arrangement of the mutations. When the two mutations were placed in the same subunit, the resulting cooperativity was neutral. In contrast, when placed in different subunits, the observed cooperativity was dominated by the mutation with strongest effects over cAMP affinity relative to wild type. These results highlight the distinct roles of intrasubunit interactions and intersubunit communication in allostery. Finally, dimers bound to either one or two cAMP molecules had similar DNA affinities, indicating that both asymmetric and symmetric liganded states activate DNA interactions. These studies have revealed the multiple communication pathways that homo-oligomers employ to transduce signals.Allosteric proteins are the basic building blocks in the transmission of biological signals, allowing communication between and within cells and from the extracellular environment to the cytosol (1). Because many allosteric proteins form homo-oligomeric complexes to modulate a ligand-induced biological response (2, 3), intersubunit communication must play a crucial role in the transduction of an allosteric signal (4, 5). Identifying the molecular mechanisms of intersubunit communication has important implications, from understanding how biological systems detect and transduce signals (6, 7) to reengineering of signaling proteins (8 -10) and to developing allosteric therapeutic modulators with enhanced affinities and specificities (11,12). Despite its importance, dissecting the mechanisms of transduction of allosteric signals from one protein subunit to another has proven difficult because it requires monitoring intermediate liganded states that are poorly populated, especially if the protein displays positive ligand binding cooperativity. Therefore, one of the remaining unresolved issues in allostery is dissecting the directionality of pathways of signal transmissions across protein subunits.A widely used s...

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Section: Quantification Of Camp Binding and Dna Interactions In Crp Scsupporting

confidence: 79%

Asymmetric configurations in a reengineered homodimer reveal multiple subunit communication pathways in protein allostery

Lanfranco

Gárate

Engdahl³

et al. 2017

Journal of Biological Chemistry

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“…We hypothesize that the activation by adenosine and the apparently improved affinity for cAMP are related, simply reflecting an improved affinity for related molecules and perhaps bringing the affinity for adenosine to the detectable level. The activation by cGMP must be different, however, because WT CRP binds cGMP with high affinity (11,24,36), so mere improvement of cGMP-binding affinity in A144T CRP cannot be the basis for the activation by cGMP. Some other CRP* variants have also been shown to be activated by GMP, at least in vivo (1,5,9).…”

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confidence: 99%

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

Youn¹,

Koh

Roberts³

2008

J Bacteriol

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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 Å) 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.The cyclic AMP (cAMP) receptor protein (CRP) of Escherichia coli is a well-studied global transcriptional regulator whose activation is highly specific to the binding of cAMP (13). In its cAMP-bound form, CRP binds DNA and interacts with RNA polymerase to stimulate transcription of appropriate genes (3, 21). In the absence of cAMP, CRP displays negligible affinity for DNA. The cAMP specificity of wild-type (WT) CRP is consistent with the structure of active CRP, which has been characterized a number of times and shows specific contacts between protein residues and cAMP (27,29,33). CRP is a homodimer in which each subunit contains two domains, the cAMP-binding domain and the DNA-binding domain, separated by a hinge region (27). The structure of the inactive form of CRP has never been determined, so the mechanism of allosteric activation by cAMP is conjectural, but plausible models for the conformational change that cAMP effects have been proposed (29,39).CRP variants have been found that have altered ligand specificity (1,5,9,14,19,23,40). The substitutions in some of these variants lie in the effector-binding pocket itself and presumably alter the specific interactions between CRP and the ligand (23, 40). Other CRP variants are fundamentally different and more difficult to explain. Specifically, there is a subset of ligandindependent (CRP*) variants that have detectable in vivo activity in the absence of cAMP and also altered ligand specificity. In these cases, the mutationally altered sites lie far from the cAMP-binding pocket, and it is therefore surprising that they alter functional properties of the ligands. A well-studied case is that of A144T CRP, which displays not only a significant activation b...

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“…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).…”

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confidence: 99%

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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Thermodynamics of Cyclic Nucleotide Binding to the cAMP Receptor Protein and Its T127L Mutant

Abstract: The thermodynamics of the binding of cyclic adenosine monophosphate (cAMP) and its non-functional analog, cyclic guanosine monophosphate (cGMP), to cyclic AMP receptor protein (CRP) and its T127L mutant were investigated by isothermal titration calorimetry (

Cited by 60 publications

References 19 publications

Asymmetric configurations in a reengineered homodimer reveal multiple subunit communication pathways in protein allostery

Asymmetric configurations in a reengineered homodimer reveal multiple subunit communication pathways in protein allostery

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

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

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