Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 83, 127 and 128 of the cyclic nucleotide binding pocket.
The cyclic 3', 5' adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli 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. Cells that expressed the binding pocket residue-substituted forms of CRP were characterized by measurements of beta-galactosidase activity. Purified wild-type and mutant CRP preparations were characterized by measurement of cAMP binding activity and by their capacity to support lacP activation in vitro. CRP structure was assessed by measurement of sensitivity to protease and DTNB-mediated subunit crosslinking. The results of this study show that cAMP interactions with serine 83, threonine 127 and serine 128 contribute to CRP activation and have little effect on cAMP binding. Amino acid substitutions that introduce hydrophobic amino acid side chain constituents at either position 127 or 128 decrease CRP discrimination of cAMP and cGMP. Finally, cAMP-induced CRP structural change(s) that occur in or near the CRP hinge region result from cAMP interaction with threonine 127; substitution of threonine 127 by cysteine, glycine, isoleucine, or serine produced forms of CRP that contained, independently of cAMP binding, structural changes similar to those of the wild-type CRP:cAMP complex.
Lee
E J
EJ
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock 79409-1061.
Glasgow
J
J
Leu
S F
SF
Belduz
A O
AO
Harman
J G
JG
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D003545:Cysteine; D004228:Dithionitrobenzoic Acid; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D005998:Glycine; D007532:Isoleucine; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011953:Receptors, Cyclic AMP / Q000235:genetics*; D012694:Serine; D013329:Structure-Activity Relationship; D013912:Threonine; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D003432:Cross-Linking Reagents; D011953:Receptors, Cyclic AMP; D007532:Isoleucine; D013912:Threonine; D012694:Serine; D004228:Dithionitrobenzoic Acid; D000242:Cyclic AMP; D001616:beta-Galactosidase; D003545:Cysteine; D005998:Glycine