Energetics of intersubunit and intrasubunit interactions of Escherichia coli adenosine cyclic 3',5'-phosphate receptor protein.


Abstract

Escherichia coli cAMP receptor protein (CRP) regulates the expression of a large number of catabolite-sensitive genes. The mechanism of CRP regulation most likely involves communication between subunits and domains. A specific message, such as the activation of CRP, may be manifested as a change in the interactions between these structural entities. Hence, the elucidation of the regulatory mechanism would require a quantitative evaluation of the energetics involved in these interactions. Thus, a study was initiated to define the conditions for reversible denaturation of CRP and to quantitatively assess the energetics involved in the intra- and intersubunit interactions in CRP. The denaturation of CRP was induced by guanidine hydrochloride. The equilibrium unfolding reaction of CRP was monitored by three spectroscopic techniques, namely, fluorescence intensity, fluorescence anisotropy, and circular dichroism. The spectroscopic data implied that CRP unfolds in a single cooperative transition. Sedimentation equilibrium data showed that CRP is dissociated into its monomeric state in high concentrations of denaturant. Unfolding of CRP is completely reversible, as indicated by fluorescence and circular dichroism measurements, and sedimentation data indicated that a dimeric structure of CRP was recovered. The functional and other structural properties of renatured and native CRP have also been examined. Quantitatively identical results were obtained. Results from additional studies as a function of protein concentration and from computer simulation demonstrated that the denaturation of CRP induced by guanidine hydrochloride proceeds according to the following pathway: (CRP2)Native<-->2(CRP)Native<-->2(CRP)Denatured. The delta G values for dissociation (delta Gd) and unfolding (delta G(u)) in the absence of guanidine hydrochloride were determined by linear extrapolation, yielding values of 12.0 +/- 0.6 and 7.2 +/- 0.1 kcal/mol, respectively. To examine the effect of the DNA binding domain on the stability of the cAMP binding domain, two proteolytically resistant cAMP binding cores were prepared from CRP in the presence of cAMP by subtilisin and chymotrypsin digestion, yielding S-CRP and CH-CRP, respectively. Results from an equilibrium denaturation study indicated that the denaturation of both CH-CRP and S-CRP is also completely reversible. Both S-CRP and CH-CRP exist as stable dimers with similar delta Gd values of 10.1 +/- 0.4 and 9.5 +/- 0.4 kcal/mol, respectively. Results from this study in conjunction with crystallographic data [McKay, D. B., Weber, I. T., & Stietz, T. A. (1982) J. Biol. Chem. 257, 9518-9524] indicate that the DNA binding domain and the C-helix are not the only structural elements that are responsible for subunit dimerization.(ABSTRACT TRUNCATED AT 400 WORDS) Study holds ProTherm entries: 4555 Extra Details: additive : EDTA(1 mM), regulatory mechanism; energetics; cooperative transition;,subunit dimerization

Submission Details

ID: QEatGfPi

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:26 p.m.

Version: 1

Publication Details
Cheng X;Gonzalez ML;Lee JC,Biochemistry (1993) Energetics of intersubunit and intrasubunit interactions of Escherichia coli adenosine cyclic 3',5'-phosphate receptor protein. PMID:8394127
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2WC2 2009-04-21 Nmr structure of catabolite activator protein in the unliganded state
4BHP 2013-05-08 A structural model of CAP mutant (T127L and S128I) in cGMP-bound state
4BH9 2013-05-08 A structural model of CAP mutant (T127L and S128I) in the apo state
4R8H 2015-07-22 1.46 The role of protein-ligand contacts in allosteric regulation of the Escherichia coli Catabolite Activator Protein
4HZF 2013-10-30 1.48 structure of the wild type Catabolite gene Activator Protein
4I0B 2013-10-30 1.5 structure of the mutant Catabolite gene activator protein H160L
3RYP 2012-05-02 1.6 Domain-domain flexibility leads to allostery within the camp receptor protein (CRP)
3KCC 2009-11-17 1.66 Crystal structure of D138L mutant of Catabolite Gene Activator Protein
4I02 2013-10-30 1.75 structure of the mutant Catabolite gene activator protein V140A
1HW5 2001-01-17 1.82 THE CAP/CRP VARIANT T127L/S128A
1I5Z 2003-06-17 1.9 STRUCTURE OF CRP-CAMP AT 1.9 A
3QOP 2012-04-04 1.96 Domain-domain flexibility leads to allostery within the camp receptor protein (CRP)
4FT8 2013-12-18 1.97 E. coli Catabolite Activator Protein with Cobalt and Sulfate Ligands
3IYD 2009-11-10 19.8 Three-dimensional EM structure of an intact activator-dependent transcription initiation complex
4I09 2013-10-30 2.05 structure of the mutant Catabolite gene activator protein V132L
1G6N 2000-12-15 2.1 2.1 ANGSTROM STRUCTURE OF CAP-CAMP
3ROU 2012-05-02 2.1 Domain-domain flexibility leads to allostery within the camp receptor protein (CRP)
1ZRF 2006-03-21 2.1 4 crystal structures of CAP-DNA with all base-pair substitutions at position 6, CAP-[6C;17G]ICAP38 DNA
4I0A 2013-10-30 2.2 structure of the mutant Catabolite gene activator protein V132A
1I6X 2003-06-17 2.2 STRUCTURE OF A STAR MUTANT CRP-CAMP AT 2.2 A
2CGP 1998-02-04 2.2 CATABOLITE GENE ACTIVATOR PROTEIN/DNA COMPLEX, ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
2GZW 2007-05-15 2.21 Crystal structure of the E.coli CRP-cAMP complex
3FWE 2009-09-08 2.3 Crystal Structure of the Apo D138L CAP mutant
4I01 2013-10-30 2.3 Structure of the mutant Catabolite gen activator protein V140L
1J59 2002-03-01 2.5 CATABOLITE GENE ACTIVATOR PROTEIN (CAP)/DNA COMPLEX + ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
3RPQ 2012-05-02 2.61 Domain-domain flexibility leads to allostery within the camp receptor protein (CRP)
1RUO 1997-01-10 2.7 CATABOLITE GENE ACTIVATOR PROTEIN (CAP) MUTANT/DNA COMPLEX + ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
3RYR 2012-05-02 2.7 Domain-domain flexibility leads to allostery within the camp receptor protein (CRP)
1RUN 1997-01-10 2.7 CATABOLITE GENE ACTIVATOR PROTEIN (CAP)/DNA COMPLEX + ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
1ZRE 2006-03-21 2.8 4 crystal structures of CAP-DNA with all base-pair substitutions at position 6, CAP-[6G;17C]ICAP38 DNA
1ZRC 2006-03-21 2.8 4 Crystal structures of CAP-DNA with all base-pair substitutions at position 6, CAP-ICAP38 DNA
1ZRD 2006-03-21 2.8 4 crystal structures of CAP-DNA with all base-pair substitutions at position 6, CAP-[6A;17T]ICAP38 DNA
1O3T 2003-04-08 2.8 PROTEIN-DNA RECOGNITION AND DNA DEFORMATION REVEALED IN CRYSTAL STRUCTURES OF CAP-DNA COMPLEXES
3RDI 2012-05-02 2.95 Domain-domain flexibility leads to allostery within the camp receptor protein (CRP)
3N4M 2011-05-25 2.99 E. coli RNA polymerase alpha subunit C-terminal domain in complex with CAP and DNA
1CGP 1994-01-31 3.0 CATABOLITE GENE ACTIVATOR PROTEIN (CAP)/DNA COMPLEX + ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE
1O3Q 2003-04-08 3.0 PROTEIN-DNA RECOGNITION AND DNA DEFORMATION REVEALED IN CRYSTAL STRUCTURES OF CAP-DNA COMPLEXES
1O3R 2003-04-08 3.0 PROTEIN-DNA RECOGNITION AND DNA DEFORMATION REVEALED IN CRYSTAL STRUCTURES OF CAP-DNA COMPLEXES
1O3S 2003-04-08 3.0 PROTEIN-DNA RECOGNITION AND DNA DEFORMATION REVEALED IN CRYSTAL STRUCTURES OF CAP-DNA COMPLEXES
1LB2 2002-09-06 3.1 Structure of the E. coli alpha C-terminal domain of RNA polymerase in complex with CAP and DNA
3HIF 2009-09-08 3.59 The crystal structure of apo wild type CAP at 3.6 A resolution.
6B6H 2017-11-15 3.9 The cryo-EM structure of a bacterial class I transcription activation complex
5CIZ 2016-06-29 5.01 E. coli RNA polymerase alpha subunit CTD in complex with CAP and DNA: A(5)-tract binding site for alpha CTD

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.5 cAMP-activated global transcriptional regulator CRP P0A2T6 CRP_SALTY
99.5 cAMP-activated global transcriptional regulator CRP P0A2T7 CRP_KLEAE
100.0 cAMP-activated global transcriptional regulator CRP P0ACK1 CRP_SHIFL
100.0 cAMP-activated global transcriptional regulator CRP P0ACJ8 CRP_ECOLI
100.0 cAMP-activated global transcriptional regulator CRP P0ACJ9 CRP_ECOL6
100.0 cAMP-activated global transcriptional regulator CRP P0ACK0 CRP_ECO57