Nucleic Acids Research, 1994, Vol. 22, No. 15 2894-2901
© 1994
MOLECULAR BIOLOGY |
Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 83, 127 and 128 the cyclic nucleotide binding pocket
Department of Chemistry and Biochemistry, Texas Tech University Lubbock, TX 79409-1061, USA
*To whom correspondence should be addressed
Received June 3, 1994. Revised June 9, 1994. Accepted June 9, 1994.
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 ß-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 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 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 wildtype CRP:cAMP complex.
+Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA 90024-1737, USA
Karadeniz Technical University, Department of Biology, 61080-Trabzon, Turkey
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Youn, J. Koh, and G. P. Roberts Two-State Allosteric Modeling Suggests Protein Equilibrium as an Integral Component for Cyclic AMP (cAMP) Specificity in the cAMP Receptor Protein of Escherichia coli J. Bacteriol., July 1, 2008; 190(13): 4532 - 4540. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. J. Moore, E. L. Mettert, and P. J. Kiley Regulation of FNR Dimerization by Subunit Charge Repulsion J. Biol. Chem., November 3, 2006; 281(44): 33268 - 33275. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Youn, R. L. Kerby, M. Conrad, and G. P. Roberts Study of Highly Constitutively Active Mutants Suggests How cAMP Activates cAMP Receptor Protein J. Biol. Chem., January 13, 2006; 281(2): 1119 - 1127. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Polit, P. Bonarek, B. Kepys, S. Kedracka-Krok, A. Gorecki, and Z. Wasylewski Kinetic Studies of cAMP-induced Allosteric Changes in Mutants T127I, S128A, and T127I/S128A of the cAMP Receptor Protein from Escherichia coli J. Biol. Chem., October 31, 2003; 278(44): 43020 - 43026. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Chen and J. C. Lee Functional Roles of Loops 3 and 4 in the Cyclic Nucleotide Binding Domain of Cyclic AMP Receptor Protein from Escherichia coli J. Biol. Chem., April 4, 2003; 278(15): 13235 - 13243. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Beatson, C. B. Whitchurch, J. L. Sargent, R. C. Levesque, and J. S. Mattick Differential Regulation of Twitching Motility and Elastase Production by Vfr in Pseudomonas aeruginosa J. Bacteriol., July 1, 2002; 184(13): 3605 - 3613. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. G. Ochoa de Alda and J. Houmard Genomic survey of cAMP and cGMP signalling components in the cyanobacterium Synechocystis PCC 6803 Microbiology, December 1, 2000; 146(12): 3183 - 3194. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Passner and T. A. Steitz The structure of a CAP-DNA complex having two cAMP molecules bound to each monomer PNAS, April 1, 1997; 94(7): 2843 - 2847. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Wang, Y. Shi, I. Gorshkova, and F. P. Schwarz RNA Polymerase-cNMP-ligated cAMP Receptor Protein (CRP) Mutant Interactions in the Enhancement of Transcription by CRP Mutants J. Biol. Chem., October 20, 2000; 275(43): 33457 - 33463. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Y. Chu, M. Tordova, G. L. Gilliland, I. Gorshkova, Y. Shi, S. Wang, and F. P. Schwarz The Structure of the T127L/S128A Mutant of cAMP Receptor Protein Facilitates Promoter Site Binding J. Biol. Chem., March 30, 2001; 276(14): 11230 - 11236. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. V. Thorsteinsson, R. L. Kerby, H. Youn, M. Conrad, J. Serate, C. R. Staples, and G. P. Roberts Redox-mediated Transcriptional Activation in a CooA Variant J. Biol. Chem., July 13, 2001; 276(29): 26807 - 26813. [Abstract] [Full Text] [PDF]
|
|