PROBING THE MECHANISM OF CRP ACTIVATION BY SITE-DIRECTED MUTAGENESIS - THE ROLE OF SERINE-128 IN THE ALLOSTERIC PATHWAY OF CAMP RECEPTOR PROTEIN-ACTIVATION

Upon activation by cAMP, Escherichia coli cAMP receptor protein (CRP) controls the expression of a network of catabolite sensitive genes. The activation of CRP by cAMP involves conformational changes such as realignments between subunits and domains within the protein. To understand the molecular events that lead to the activation of CRP, point mutations at position 128 were introduced via site-directed mutagenesis in an attempt to specifically affect the subunit interfacial interactions, as well as the Ligand-binding reaction. The biochemical and biophysical properties of these mutants were rigorously tested with the goal of identifying the partial reactions in the activation pathway that are perturbed by this specific mutation. Results from this study suggest that mutation of Ser 128 to Ala or Pro does not significantly disturb the overall secondary structure as monitored by circular dichroism. The energetics of subunit-subunit interaction and protein stability were monitored by sedimentation and spectroscopic techniques. Although these mutants were designed to interrupt intersubunit interactions, the energetics of subunit association and protein stability remain quantitatively the same as those of the wild-type CRP. Nevertheless, the ability of the subunit to be realigned to the DNA-binding form is significantly affected as reflected by the pronounced decrease in the susceptibility of mutant CRP to proteolytic digestion in the presence of cAMP. In addition, the binding affinity of cAMP to the first ligand site in mutants S128A and S128P is the same as that of the wild type, but the affinity to the second ligand site is reduced. This observation indicates that mutation at position 128 affects ligand binding by amplifying the magnitude of negative cooperativity. Mutation at residue 128 does not impair the ability of interdomain interactions as indicated by the quantitative response of a spectroscopic probe in the DNA-binding domain to the binding of cAMP to the Ligand-binding domain. The S128A mutant binds to a specific DNA sequence about 50-fold weaker than the wild-type CRP, while the mutant S128P has no measurable DNA affinity under the same conditions. This observation is consistent with the in vivo result that both mutants display an inactive CRP phenotype (CRP(-)). In summary, these results suggest that communication between domains induced by cAMP binding can be dissociated from the proper subunit realignment of the CRP dimer that is crucial for the activation of CRP. Apparently, serine 128 is not vital for interdomainn communication, but plays an important role in mediating the interactions between the two subunits and discriminating between cAMP and cGMP.