Ability of E. coli Cyclic AMP receptor protein to differentiate cyclic nucelotides: Effects of single site mutations

Shwu Hwa Lin, Lubomir Kovac, Anita J. Chin, Christopher C Q Chin, James Lee

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Escherichia coli cyclic AMP receptor protein (CRP) is a global transcriptional regulator which controls the expression of many different genes. Although different cyclic nucleotides can bind to CRP with almost equal affinity, only in the presence of cAMP could wild-type CRP bind to specific DNA sequences. Molecular genetic studies have identified a class of mutants, CRP*, which either do not require exogenous cAMP for activation or can be activated by cGMP. Thus, these mutants might aid in identifying the structural elements that are involved in the modulation of CRP to correctly differentiate the messages embedded in cyclic nucleotides. In this in vitro study, five CRP* mutants, namely, D53H, S62F, G141Q, G141K, and L148R, were tested for their abilities to bind the lac promoter sequence and the effects of cyclic nucleotides in modulating DNA sequence recognition. For comparison, non-CRP* mutants K52N, T127L, H159L, and K52N/H159L were studied. cCMP and cGMP can replace cAMP as an allosteric effector in all of these CRP mutants except S62F and non-CRP* mutants. The D53H, G141Q, G141K, and L148R mutants exhibit significantly higher affinity for the lac promoter sequence than wild-type CRP while S62F and the non-CRP* mutants exhibit reduced affinity. To probe the pathway of communication, the energetics of subunit assembly in these mutants were monitored by sedimentation equilibrium, and the conformational states of these mutants were probed by proteolysis and accessibility of Cys178 to chemical modifications. Results from these studies imply that signals due to mutations are mostly transmitted through the subunit interface. Thus, residues in CRP outside of the cyclic nucleotide binding site modulate the ability of CRP to differentiate these three cyclic nucleotides through long-range communication. Furthermore, this study shows that CRP* mutations do not impart any unique properties to CRP except that the DNA binding constants are shifted to a regime of higher affinity.

Original languageEnglish (US)
Pages (from-to)2946-2955
Number of pages10
JournalBiochemistry
Volume41
Issue number9
DOIs
StatePublished - Mar 5 2002

Fingerprint

Cyclic AMP Receptor Protein
Mutation
Cyclic Nucleotides
Mutant Proteins
Adenosine Monophosphate
DNA sequences
E coli cyclic AMP receptor protein
Proteolysis
Escherichia coli Proteins
Communication
Chemical modification
Sedimentation
Escherichia coli

ASJC Scopus subject areas

  • Biochemistry

Cite this

Ability of E. coli Cyclic AMP receptor protein to differentiate cyclic nucelotides : Effects of single site mutations. / Lin, Shwu Hwa; Kovac, Lubomir; Chin, Anita J.; Chin, Christopher C Q; Lee, James.

In: Biochemistry, Vol. 41, No. 9, 05.03.2002, p. 2946-2955.

Research output: Contribution to journalArticle

Lin, Shwu Hwa ; Kovac, Lubomir ; Chin, Anita J. ; Chin, Christopher C Q ; Lee, James. / Ability of E. coli Cyclic AMP receptor protein to differentiate cyclic nucelotides : Effects of single site mutations. In: Biochemistry. 2002 ; Vol. 41, No. 9. pp. 2946-2955.
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abstract = "Escherichia coli cyclic AMP receptor protein (CRP) is a global transcriptional regulator which controls the expression of many different genes. Although different cyclic nucleotides can bind to CRP with almost equal affinity, only in the presence of cAMP could wild-type CRP bind to specific DNA sequences. Molecular genetic studies have identified a class of mutants, CRP*, which either do not require exogenous cAMP for activation or can be activated by cGMP. Thus, these mutants might aid in identifying the structural elements that are involved in the modulation of CRP to correctly differentiate the messages embedded in cyclic nucleotides. In this in vitro study, five CRP* mutants, namely, D53H, S62F, G141Q, G141K, and L148R, were tested for their abilities to bind the lac promoter sequence and the effects of cyclic nucleotides in modulating DNA sequence recognition. For comparison, non-CRP* mutants K52N, T127L, H159L, and K52N/H159L were studied. cCMP and cGMP can replace cAMP as an allosteric effector in all of these CRP mutants except S62F and non-CRP* mutants. The D53H, G141Q, G141K, and L148R mutants exhibit significantly higher affinity for the lac promoter sequence than wild-type CRP while S62F and the non-CRP* mutants exhibit reduced affinity. To probe the pathway of communication, the energetics of subunit assembly in these mutants were monitored by sedimentation equilibrium, and the conformational states of these mutants were probed by proteolysis and accessibility of Cys178 to chemical modifications. Results from these studies imply that signals due to mutations are mostly transmitted through the subunit interface. Thus, residues in CRP outside of the cyclic nucleotide binding site modulate the ability of CRP to differentiate these three cyclic nucleotides through long-range communication. Furthermore, this study shows that CRP* mutations do not impart any unique properties to CRP except that the DNA binding constants are shifted to a regime of higher affinity.",
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