Effect of cAMP binding site mutations on the interaction of cAMP receptor protein with cyclic nucleoside monophosphate ligands and DNA.
Although cAMP binding to wild type cAMP receptor protein (CRP) induces specific DNA binding and activates transcription, cyclic nucleoside monophosphate (cNMP) binding to the CRP mutant Ser128 --> Ala does not, whereas the double CRP mutant Thr127 --> Leu/Ser128 --> Ala activates transcription even in the absence of cNMP. Isothermal titration calorimetry measurements on the cNMP binding reactions to the S128A and T127L/S128A mutants show that the reactions are mainly entropically driven as is cAMP binding to CRP. In contrast to cAMP binding to CRP, the binding reactions are noncooperative and exothermic with binding enthalpies (DeltaHb) ranging from -23.4 +/- 0.9 kJ mol-1 for cAMP binding to S128A at 39 degrees C to -4.1 +/- 0.6 kJ mol-1 for cAMP binding to T127L/S128A at 24 degrees C and exhibit enthalpy-entropy compensation. To account for the inactivity of the S128A mutant, in vitro and in vivo DNA binding experiments were performed on the cAMP-ligated S128A mutant. The cAMP-ligated S128A mutant binds to the consensus DNA binding site with approximately the same affinity as that of cAMP-ligated CRP but forms a different type of complex, which may account for loss of transcriptional activity by the mutant. Energy minimization computations on the cAMP-ligated S128A mutant show that amino acid conformational differences between S128A and CRP occur at Ser179, Glu181, and Thr182 in the center of the DNA binding site, implying that these conformational changes may account for the difference in DNA binding.
Moore
J L
JL
Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, Rockville, Maryland 20850, USA.
Gorshkova
I I
II
Brown
J W
JW
McKenney
K H
KH
Schwarz
F P
FP
D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism*; D002373:Cyclic AMP Receptor Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D008024:Ligands; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D013816:Thermodynamics
D002373:Cyclic AMP Receptor Protein; D004268:DNA-Binding Proteins; D008024:Ligands; D004247:DNA; D000242:Cyclic AMP; D012321:DNA-Directed RNA Polymerases