Kinetic and Structural Studies of the Allosteric Conformational Changes Induced by Binding of cAMP to the cAMP Receptor Protein from Escherichia coli

Fic, Ewelina; Polit, Agnieszka; Wasylewski, Zygmunt

doi:10.1021/bi051586a

Cited by 10 publications

(8 citation statements)

References 19 publications

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“…Therefore, it seems that two conformational states of CRP exist and only one of those states is able to bind the ligand. The obtained results suggest that the kinetic mechanism of binding of cAMP to both syn sites of CRP is very similar [Fic et al, 2006].…”

Section: Cyclic Amp Binding By Crpmentioning

confidence: 67%

“…This process can be observed by the conformational changes of CRP that surround the tryptophan residue in position 85 present in both subunits. Recently, the kinetics of cAMP binding to a CRP heterodimer mutant, in which a single Trp85 residue was present in only one subunit of the protein (CRPW85-CRP), were measured under pseudo-first-order conditions and the experimental data could be fitted to single-exponential curves [Fic et al, 2006]. The observed rate constant for the CRPW85-CRP heterodimer decreased with an increase in cAMP concentration, as observed in the case of wild-type CRP [Malecki et al, 2000].…”

Section: Cyclic Amp Binding By Crpmentioning

confidence: 99%

“…Those results proved the appearance of structural changes resulting probably from the rearrangement of the N-and C-terminal domains. Presumably, in solution both CRP subunits exist in the open conformation and after cAMP binding, there is the possibility of conversion to the closed conformation, in which both domains that form the subunit lie closer to each other [Fic et al, 2006;Polit et al, 2003a].…”

Section: Cyclic Amp Binding By Crpmentioning

confidence: 99%

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cAMP Receptor Protein from Escherichia coli as a Model of Signal Transduction in Proteins – A Review

Fic¹,

Bonarek²,

Górecki³

et al. 2008

Microb Physiol

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In Escherichia coli, cyclic AMP receptor protein (CRP) is known to regulate the transcription of about 100 genes. The signal to activate CRP is the binding of cyclic AMP. It has been suggested that binding of cAMP to CRP leads to a long-distance signal transduction from the N-terminal cAMP-binding domain to the C-terminal domain of the protein, which is responsible for interaction with specific sequences of DNA. The signal transduction plays a crucial role in the activation of the protein. The most sophisticated spectroscopic techniques, other techniques frequently used in structural biochemistry, and site-directed mutagenesis have been used to investigate the details of cAMP-mediated allosteric control over CRP conformation and activity as a transcription factor. The aim of this review is to summarize recent works and developments pertaining to cAMP-dependent CRP signal transduction in E. coli.

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Section: Cyclic Amp Binding By Crpmentioning

confidence: 67%

Section: Cyclic Amp Binding By Crpmentioning

confidence: 99%

Section: Cyclic Amp Binding By Crpmentioning

confidence: 99%

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cAMP Receptor Protein from Escherichia coli as a Model of Signal Transduction in Proteins – A Review

Fic¹,

Bonarek²,

Górecki³

et al. 2008

Microb Physiol

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“…Recently, the x-ray structures of oxidized CprK1 from D. hafniense in complex with CHPA and reduced CprK from D. dehalogenans in its unliganded form have been determined by Joyce et al (13). They reveal that both CprKs exhibit high structural similarity to the cAMP receptor protein (CRP) (14,15) and PrfA, a key virulence regulator of Listeria monocytogenes (16). Two identical subunits form an asymmetric dimer with each monomer folded in two distinct domains: the N-terminal effector binding domain and the C-terminal DNA binding domain.…”

mentioning

confidence: 99%

Transcriptional Activation by CprK1 Is Regulated by Protein Structural Changes Induced by Effector Binding and Redox State

Mazon

Gábor

Leys

et al. 2007

Journal of Biological Chemistry

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The transcriptional activator CprK1 from Desulfitobacteriumhafniense, a member of the ubiquitous cAMP receptor protein/ fumarate nitrate reduction regulatory protein family, activates transcription of genes encoding proteins involved in reductive dehalogenation of chlorinated aromatic compounds. 3-Chloro-4-hydroxyphenylacetate is a known effector for CprK1, which interacts tightly with the protein, and induces binding to a specific DNA sequence ("dehalobox," TTAAT--ATTAA) located in the promoter region of chlorophenol reductive dehalogenase genes. Despite the availability of recent x-ray structures of two CprK proteins in distinct states, the mechanism by which CprK1 activates transcription is poorly understood. In the present study, we have investigated the mechanism of CprK1 activation and its effector specificity. By using macromolecular native mass spectrometry and DNA binding assays, analogues of 3-chloro-4-hydroxyphenylacetate that have a halogenated group at the ortho position and a chloride or acetic acid group at the para position were found to be potent effectors for CprK1. By using limited proteolysis it was demonstrated that CprK1 requires a cascade of structural events to interact with dehalobox dsDNA. Upon reduction of the intermolecular disulfide bridge in oxidized CprK1, the protein becomes more dynamic, but this alone is not sufficient for DNA binding. Activation of CprK1 is a typical example of allosteric regulation; the binding of a potent effector molecule to reduced CprK1 induces local changes in the N-terminal effector binding domain, which subsequently may lead to changes in the hinge region and as such to structural changes in the DNA binding domain that are required for specific DNA binding.Halogenated hydrocarbons are often toxic molecules. They are widespread environmental pollutants because of their numerous applications, for example in industry (degreasers), agriculture (pesticides), and private households (flame retardants). Although these compounds are generally very stable, they can be converted in many sediments and soils by reductive dehalogenation. Several strictly anaerobic bacteria capable of dehalogenation have been isolated including Desulfomonile, Dehalobacter, and Desulfitobacterium. These organisms use chlorinated compounds as terminal electron acceptors (halorespiration) and thus remove the chloride atom while energy is conserved via electron transport phosphorylation (1-3). The prospect of using these organisms in bioremediation of sediments contaminated with a variety of chlorinated aromatic compounds is promising. Desulfitobacterium dehalogenans can reductively dechlorinate phenolic compounds at the ortho position (4, 5), and the closely related Desulfitobacterium hafniense DCB-2 (6) can dechlorinate phenolic compounds at the ortho and meta position; the latter reaction, however, is described only for 3,5-dichlorophenol (3,5-DCP) 2 as a substrate (7).A large number of the proteins involved in halorespiration are encoded in the chlorophenol reductive dehalogenase (cpr...

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“…Thus, the binding of CAP to specific DNA under the present experimental conditions involves some allosteric conformation changes of the protein and also some cAMP binding. The kinetics of CAPconformation changes induced by cAMP binding has been studied for various mutants and using different indicators, showing ''at least a three-step conformation change'' with reaction times extending into the range of a few seconds (Fic et al 2006(Fic et al , 2009Gorecki et al 2009). Thus, slow conformation changes of the protein are expected to slow down the reaction of CAP with promoter DNA.…”

Section: Kinetics Of Receptor Bindingmentioning

confidence: 99%

Structures during binding of cAMP receptor to promoter DNA: promoter search slowed by non-specific sites

Pörschke

2012

Eur Biophys J

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The kinetics of cAMP receptor (CAP) binding to promoter DNA has been studied by stopped-flow electric-dichroism at a reduced salt concentration, where the coupling of non-specific and specific binding can be observed directly. Amplitudes, rise and decay times of dichroism transients provide detailed information about the reaction and the structure of intermediates over more than six orders of magnitude on the time scale. CAP binding during the first milliseconds after mixing is indicated by an increase of both rise- and decay-time constants. A particularly large increase of rise times reflects initial formation of non-symmetric complexes by protein binding to non-specific sites at DNA ends. The increase of the hydrodynamic dimensions continues up to ~1 s, before a decrease of time constants reflects transition to compact states with bent DNA up to the time range of ~10(3) s. The slow approach to CAP-induced DNA bending is due to non-specific complexes, which are formed initially and are converted slowly to the specific complex. At the salt concentration of 13.5 mM, conversion to specific complexes with bent DNA is completed after ~40 s at pH 8 compared to >10(3) s at pH 7, resulting from a higher affinity of CAP to non-specific sites at pH 7 than 8 by a factor of ~100. Thus, under the given conditions non-specific sites delay rather than facilitate formation of the specific complex with bent DNA. Experimental data obtained for a non-specific DNA clearly indicate the impact of pseudo-sites. The different electro-optical parameters have been combined in global fits.

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Kinetic and Structural Studies of the Allosteric Conformational Changes Induced by Binding of cAMP to the cAMP Receptor Protein from Escherichia coli

Cited by 10 publications

References 19 publications

cAMP Receptor Protein from <i>Escherichia coli</i> as a Model of Signal Transduction in Proteins – A Review

cAMP Receptor Protein from <i>Escherichia coli</i> as a Model of Signal Transduction in Proteins – A Review

Transcriptional Activation by CprK1 Is Regulated by Protein Structural Changes Induced by Effector Binding and Redox State

Structures during binding of cAMP receptor to promoter DNA: promoter search slowed by non-specific sites

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