INTERACTIVE AND DOMINANT EFFECTS OF RESIDUE-128 AND RESIDUE-141 ON CYCLIC-NUCLEOTIDE AND DNA BINDINGS IN ESCHERICHIA-COLI CAMP RECEPTOR PROTEIN

The molecular events in the cAMP-induced allosteric activation of cAMP receptor protein (CRP) involve interfacial communications between sub units and domains. However, the roles of intersubunit and interdomain interactions in defining the selectivity of cAMP against other cyclic nucleotides and cooperativity in ligand binding are still not known, N atural occurring CRP mutants with different phenotypes were employed t o address these issues. Thermodynamic analyses of subunit association, protein stability, and cAMP and DNA binding as well as conformational studies of the mutants and wild-type CRPs lead to an identification o f the apparently dominant roles of residues 128 and 141 in the cAMP-mo dulated DNA binding activity of CRP, Serine 128 and the C-helix were i mplicated as playing a critical role in modulating negative cooperativ ity of cyclic nucleotide binding. A correlation was established betwee n a weak affinity for subunit assembly and the relaxation of cyclic nu cleotide selectivity in the G141Q and S128A/G141Q mutants, These resul ts imply that intersubunit interaction is important for cyclic nucleot ide discrimination in CRP, The double mutant S128A/G141Q, constructed from two single mutations of S128A and G141Q, which exhibit opposite p henotypic characteristics of CRP- and CRP, respectively, assumes a CR P phenotype and has biochemical properties similar to those of the G1 41Q mutant. These observations suggest that mutation G141Q exerts a do minant effect over mutation S128A and that the subunit realignment ind uced by the G141Q mutation can override the local structural disruptio n created by mutation S128A.