Arabinose Alters Both Local and Distal H-D Exchange Rates in the Escherichia coli AraC Transcriptional Regulator.

In the absence of arabinose, the dimeric Escherichia coli regulatory protein of the L-arabinose operon, AraC, represses expression by looping the DNA between distant half-sites. Arabinose binding to the dimerization domains transition AraC to preferentially bind two adjacent DNA half-sites, which stimulates RNA polymerase transcription of the araBAD catabolism genes. Prior genetic and biochemical studies hypothesize that arabinose allosterically induces a helix-coil transition of a linker between the dimerization and DNA binding domains that switches the AraC conformation to an inducing state [1]. To test this hypothesis, hydrogen-deuterium exchange mass spectrometry (HXMS) was utilized to identify structural regions involved in the conformational activation of AraC by arabinose. Comparison of the hydrogen-deuterium exchange kinetics of individual dimeric dimerization domains and the full-length dimeric AraC protein in the presence and absence of arabinose reveals a prominent arabinose-induced destabilization of the amide hydrogen bonded structure of linker residues (I_167-N_168). This destabilization is demonstrated to result from an increased probability to form a helix capping motif at the C-terminal end of the dimerizing $\alpha$-helix of the dimerization domain that preceeds the interdomain linker. These conformational changes could allow for quaternary repositioning of the DNA binding domains required for induction of the araBAD promoter through rotation of peptide backbone dihedral angles of just a couple residues. Subtle changes in exchange rates are also visible around the arabinose binding pocket and in the DNA binding domain.