Interactive and dominant effects of residues 128 and 141 on cyclic nucleotide and DNA bindings in Escherichia coli cAMP receptor protein

Xiaodong Cheng, James Lee

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

The molecular events in the cAMP-induced allosteric activation of cAMP receptor protein (CRP) involve interfacial communications between subunits and domains. However, the roles of intersubunit and interdomain interactions in defining the selectivity of cAMP against other cyclic nucleotides and cooperativity in ligand binding are still not known. Natural occurring CRP mutants with different phenotypes were employed to address these issues. Thermodynamic analyses of subunit association, protein stability, and cAMP and DNA binding as well as conformational studies of the mutants and wild- type CRPs lead to an identification of the apparently dominant roles of residues 128 and 141 in the cAMP-modulated DNA binding activity of CRP. Serine 128 and the C-helix were implicated as playing a critical role in modulating negative cooperativity of cyclic nucleotide binding. A correlation was established between a weak affinity for subunit assembly and the relaxation of cyclic nucleotide selectivity in the G141Q and S128A/ G141Q mutants. These results imply that intersubunit interaction is important for cyclic nucleotide discrimination in CRP. The double mutant S128A/G141Q, constructed from two single mutations of S128A and G141Q, which exhibit opposite phenotypic characteristics of CRP- and CRP+, respectively, assumes a CRP+ phenotype and has biochemical properties similar to those of the G141Q mutant. These observations suggest that mutation G141Q exerts a dominant effect over mutation S128A and that the subunit realignment induced by the G141Q mutation can override the local structural disruption created by mutation S128A.

Original languageEnglish (US)
Pages (from-to)705-712
Number of pages8
JournalJournal of Biological Chemistry
Volume273
Issue number2
DOIs
StatePublished - Jan 9 1998

Fingerprint

Cyclic AMP Receptor Protein
Escherichia coli Proteins
Cyclic Nucleotides
Escherichia coli
DNA
Mutation
Phenotype
Protein Stability
Thermodynamics
Serine
Chemical activation
Association reactions
Ligands
Communication

ASJC Scopus subject areas

  • Biochemistry

Cite this

Interactive and dominant effects of residues 128 and 141 on cyclic nucleotide and DNA bindings in Escherichia coli cAMP receptor protein. / Cheng, Xiaodong; Lee, James.

In: Journal of Biological Chemistry, Vol. 273, No. 2, 09.01.1998, p. 705-712.

Research output: Contribution to journalArticle

@article{fea74db64a074b94897e8766e2419ad6,
title = "Interactive and dominant effects of residues 128 and 141 on cyclic nucleotide and DNA bindings in Escherichia coli cAMP receptor protein",
abstract = "The molecular events in the cAMP-induced allosteric activation of cAMP receptor protein (CRP) involve interfacial communications between subunits and domains. However, the roles of intersubunit and interdomain interactions in defining the selectivity of cAMP against other cyclic nucleotides and cooperativity in ligand binding are still not known. Natural occurring CRP mutants with different phenotypes were employed to address these issues. Thermodynamic analyses of subunit association, protein stability, and cAMP and DNA binding as well as conformational studies of the mutants and wild- type CRPs lead to an identification of the apparently dominant roles of residues 128 and 141 in the cAMP-modulated DNA binding activity of CRP. Serine 128 and the C-helix were implicated as playing a critical role in modulating negative cooperativity of cyclic nucleotide binding. A correlation was established between a weak affinity for subunit assembly and the relaxation of cyclic nucleotide selectivity in the G141Q and S128A/ G141Q mutants. These results imply that intersubunit interaction is important for cyclic nucleotide discrimination in CRP. The double mutant S128A/G141Q, constructed from two single mutations of S128A and G141Q, which exhibit opposite phenotypic characteristics of CRP- and CRP+, respectively, assumes a CRP+ phenotype and has biochemical properties similar to those of the G141Q mutant. These observations suggest that mutation G141Q exerts a dominant effect over mutation S128A and that the subunit realignment induced by the G141Q mutation can override the local structural disruption created by mutation S128A.",
author = "Xiaodong Cheng and James Lee",
year = "1998",
month = "1",
day = "9",
doi = "10.1074/jbc.273.2.705",
language = "English (US)",
volume = "273",
pages = "705--712",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "2",

}

TY - JOUR

T1 - Interactive and dominant effects of residues 128 and 141 on cyclic nucleotide and DNA bindings in Escherichia coli cAMP receptor protein

AU - Cheng, Xiaodong

AU - Lee, James

PY - 1998/1/9

Y1 - 1998/1/9

N2 - The molecular events in the cAMP-induced allosteric activation of cAMP receptor protein (CRP) involve interfacial communications between subunits and domains. However, the roles of intersubunit and interdomain interactions in defining the selectivity of cAMP against other cyclic nucleotides and cooperativity in ligand binding are still not known. Natural occurring CRP mutants with different phenotypes were employed to address these issues. Thermodynamic analyses of subunit association, protein stability, and cAMP and DNA binding as well as conformational studies of the mutants and wild- type CRPs lead to an identification of the apparently dominant roles of residues 128 and 141 in the cAMP-modulated DNA binding activity of CRP. Serine 128 and the C-helix were implicated as playing a critical role in modulating negative cooperativity of cyclic nucleotide binding. A correlation was established between a weak affinity for subunit assembly and the relaxation of cyclic nucleotide selectivity in the G141Q and S128A/ G141Q mutants. These results imply that intersubunit interaction is important for cyclic nucleotide discrimination in CRP. The double mutant S128A/G141Q, constructed from two single mutations of S128A and G141Q, which exhibit opposite phenotypic characteristics of CRP- and CRP+, respectively, assumes a CRP+ phenotype and has biochemical properties similar to those of the G141Q mutant. These observations suggest that mutation G141Q exerts a dominant effect over mutation S128A and that the subunit realignment induced by the G141Q mutation can override the local structural disruption created by mutation S128A.

AB - The molecular events in the cAMP-induced allosteric activation of cAMP receptor protein (CRP) involve interfacial communications between subunits and domains. However, the roles of intersubunit and interdomain interactions in defining the selectivity of cAMP against other cyclic nucleotides and cooperativity in ligand binding are still not known. Natural occurring CRP mutants with different phenotypes were employed to address these issues. Thermodynamic analyses of subunit association, protein stability, and cAMP and DNA binding as well as conformational studies of the mutants and wild- type CRPs lead to an identification of the apparently dominant roles of residues 128 and 141 in the cAMP-modulated DNA binding activity of CRP. Serine 128 and the C-helix were implicated as playing a critical role in modulating negative cooperativity of cyclic nucleotide binding. A correlation was established between a weak affinity for subunit assembly and the relaxation of cyclic nucleotide selectivity in the G141Q and S128A/ G141Q mutants. These results imply that intersubunit interaction is important for cyclic nucleotide discrimination in CRP. The double mutant S128A/G141Q, constructed from two single mutations of S128A and G141Q, which exhibit opposite phenotypic characteristics of CRP- and CRP+, respectively, assumes a CRP+ phenotype and has biochemical properties similar to those of the G141Q mutant. These observations suggest that mutation G141Q exerts a dominant effect over mutation S128A and that the subunit realignment induced by the G141Q mutation can override the local structural disruption created by mutation S128A.

UR - http://www.scopus.com/inward/record.url?scp=2642612817&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=2642612817&partnerID=8YFLogxK

U2 - 10.1074/jbc.273.2.705

DO - 10.1074/jbc.273.2.705

M3 - Article

C2 - 9422721

AN - SCOPUS:2642612817

VL - 273

SP - 705

EP - 712

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 2

ER -