Proposed structural mechanism of Escherichia coli cAMP receptor protein cAMP-dependent proteolytic cleavage protection and selective and nonselective DNA binding.
The Escherichia coli cAMP receptor protein (CRP) displays biphasic characteristics in protease and beta-galactosidase induction assays at increasing cAMP concentrations in response to ligand binding at the secondary binding site located between the primary binding site and the DNA binding domain. Two mutants were created to determine the mechanistic reason for the CRP biphasic response by inhibiting binding of cAMP to the secondary site via interference with the Arg 181 interaction with the ligand's phosphate. The S179A/R180D/E181H mutant binds two cAMP molecules per dimer, does not exhibit a biphasic response, lacks selective DNA binding, and has inhibited nonselective DNA binding. The R180K mutant binds four cAMP molecules per dimer, exhibits a biphasic response, nonselective DNA binding similar to CRP, but has inhibited selective DNA binding characteristics. The results are consistent with a 2 x 2-binding site scheme were both primary binding sites must be occupied before the secondary binding sites are occupied. A structural mechanism suggesting the secondary sites are formed by binding of cAMP to the primary sites is proposed. AMMP-generated molecular models suggest that R180 orients E181 to produce selective DNA binding, Arg 169 interactions are necessary for nonselective DNA binding, and the position of Leu 57 inhibits chymotrypsin cleavage of Phe 136. DNA binding results suggest that CRP may be the unknown transcription factor which binds to the temperature sensitive dsrA promoter.
Scott
Sean-Patrick
SP
Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204, USA. spscott@uh.edu
Jarjous
Shadi
S
D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D000242:Cyclic AMP / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry* / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D017433:Protein Structure, Secondary
D002373:Cyclic AMP Receptor Protein; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D000242:Cyclic AMP; D006152:Cyclic GMP