Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: Targeting positions 83, 127, and 128 of the cyclic nucleotide binding pocket

Date
1993-12
Journal Title
Journal ISSN
Volume Title
Publisher
Texas Tech University
Abstract

The cyclic 3':5'-adenosine monophosphate (cAMP) receptor protein (CRP) of Escherichia coli binds both cyclic nucleotides and specific sequences in several promoter regions. The activation of CRP as a transcriptional control element involves cAMP binding and cAMP-mediated allosteric change of CRP structure. Five specific amino acid residues are predicted to play a role in the interaction of cAMP with CRP. The purpose of this work was to test the role of three amino acid residues, serine at position 83, threonine at position 127 and serine at position 128, in facilitating both cAMP binding and cAMP-mediated activation of CRP. The cAMP binding pocket of CRP was mutagenized to substitute cysteine or glycine for serine 83; cysteine, glycine, isoleucine, or serine for threonine 127; and threonine or alanine for serine 128. Substitutions were made in wild-type CRP and in a CRP*, or cAMP-independent, form of the protein to assess the effects of the amino acid substitutions on CRP structure. Cells that expressed the binding pocket residue-substituted forms of CRP were characterized through measurement of p-galactosidase activity. Purified wild-type and mutant CRP preparations were characterized by measurement of cAMP binding activity, and their structures were assessed by measurement of protease sensitivity and DTNBmediated subunit crosslinking. The results of this study show that cAMP interactions with serine 83, threonine 127 and serine 128 contribute to CRP activation and little effect on cAMP binding. Serine 128 and threonine 127 were found to play a role in CRP discrimination of cAMP and cGMP. cAMPinduced CRP structural change(s) that occur in or near the CRP hinge region appear to result from cAMP interaction with threonine 127; substitution of threonine 127 by either cysteine, glycine, isoleucine, or serine, produced similar structural changes in CRP independently of cAMP binding.

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Keywords
Escherichia coli -- Genetic engineering, Serine proteinases -- Analysis, Radioligand assay, Adenine nucleotides, Cyclic adenylic acid -- Analysis
Citation