Characterization of the oligomeric states of wild type and mutant AraC

AraC regulates transcription of the Escherichia coli arabinose operon, binding tandem DNA half-sites in the presence of arabinose and widely spaced half-sites in the absence of arabinose. In the structure of the AraC N-terminal dimerization domain with bound arabinose, the protein dimerizes via an antiparallel coiled-coil interface. The absence of bound ligand opens a second, beta-barrel interaction interface that also mediates interactions between unliganded AraC dimers in the crystal. The larger buried surface area of the beta-barrel interface, as compared with the coiled-coil interface, raised the possibility that protein-protein interactions mediated by the beta-barrel might play a role in ligand-mediated modulation of AraC DNA binding activity. For the crystallographically observed beta-barrel interaction to play a role in the cell, dimerization via this interface in the absence of arabinose would be predicted to be at least as energetically favorable as dimerization via the coiled-coil interface. In the study presented here, we use analytical ultracentrifugation to determine the oligomeric state of the AraC dimerization domain in the presence and absence of arabinose. Dimerization of the unliganded protein via the beta-barrel interface in the absence of interactions mediated by the coiled-coil interface is assayed using a mutant AraC protein with a disrupted coiled-coil interface. The results of these studies indicate that dimerization via the beta-barrel interface is substantially weaker than dimerization via the coiled-coil interface, indicating that the crystallographically observed beta-barrel interaction is not relevant to in vivo function.