Determination of the conformations of cAMP receptor protein and its T127L,S128A mutant with and without cAMP from small angle neutron scattering measurements.

Small angle neutron scattering (SANS) measurements were performed on solutions of cAMP receptor protein (CRP) and on solutions of the T127L,S128A double mutant of CRP (CRP*) in D2O K3PO4 buffer containing 0.5 M KCl, in the absence and presence of 3',5' cyclic adenosine monophosphate (cAMP). Energy-minimized structures of the CRP were calculated by minimization of the x-ray crystallographic structure of CRP in either the exclusively "closed" form where the alpha-helices of the carboxyl-terminal domain are folded close to the amino-terminal domain and in the exclusively "open" form where the alpha-helices of the carboxyl-terminal domain are folded away from the amino-terminal domain. Neutron scattering models show that the CRP SANS data follow closely the data curve predicted for unligated CRP in the open form, whereas the cAMP-ligated data are more in agreement with the data predicted for the minimized cAMP-ligated CRP structure in the closed form. Thus, it appears that CRP undergoes a conformational change from the open form to the closed form in solution upon ligation with cAMP. The SANS data from the CRP* and cAMP-ligated CRP* are coincidental, which implies that there is very little structural difference between the two species of CRP*. This is in agreement with in vivo results, which show that whereas CRP activates transcription in the cell only in the presence of cAMP, CRP* activates transcription in the absence of cAMP, implying that CRP* is already in the correct conformation for the activation of transcription.

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.

Thermodynamics of cyclic nucleotide binding to the cAMP receptor protein and its T127L mutant.

The thermodynamics of the binding of cyclic adenosine monophosphate (cAMP) and its non-functional analog, cyclic guanosine monophosphate (cGMP), to cyclic AMP receptor protein (CRP) and its T127L mutant were investigated by isothermal titration calorimetry (ITC) in 0.2 and 0.5 M KCl phosphate buffer (pH 7.0) at 24 and 39 degrees C. Although, the binding of the first cAMP molecule to CRP is exothermic with an enthalpy change (delta Hb) of -6 kJ mol-1, a heat capacity change (delta Cp) of -0.300 kJ mol-1 K-1, and an entropy increase (delta Sb) of 72 J mol-1 K-1, the overall binding of cAMP to CRP is endothermic and positively cooperative: binding of the first cAMP molecule increases the affinity for the second one by more than an order of magnitude at 24 degrees C. The binding of the second cAMP molecule is accompanied by large changes of 48.1 kJ mol-1 in delta Hb, of -1.4 kJ mol-1 K-1 in delta Cp, and of 255 J mol-1 K-1 in delta Sb at 24 degrees C and 0.5 M KCl phosphate buffer. In contrast, the overall binding of cGMP to CRP is exothermic and non-cooperative with delta Hb, delta Cp, and delta Sb values close to the those values for binding of the first cAMP molecule to CRP. The point mutation, T127L, switches off the cooperativity between the cAMP ligated binding sites without affecting the binding constant of cAMP and changes the specificity of the protein so that transcription is now activated only upon cGMP binding. All the binding reactions to CRP and the mutant are mainly entropically driven at 24 degrees C.