bi9b00389_si_001.pdf (11.36 MB)
Arabinose Alters Both Local and Distal H–D Exchange Rates in the Escherichia coli AraC Transcriptional Regulator
journal contributionposted on 2019-06-14, 00:00 authored by Alexander Tischer, Matthew J. Brown, Robert F. Schleif, Matthew Auton
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. Binding of arabinose to the dimerization domains forces AraC to preferentially bind two adjacent DNA half-sites, which stimulates RNA polymerase transcription of the araBAD catabolism genes. Prior genetic and biochemical studies hypothesized 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 [Brown, M. J., and Schleif, R. F. (2019) Biochemistry, preceding paper in this issue (DOI: 10.1021/acs.biochem.9b00234)]. To test this hypothesis, hydrogen–deuterium exchange mass spectrometry 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 (I167 and N168). This destabilization is demonstrated to result from an increased probability to form a helix capping motif at the C-terminal end of the dimerizing α-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 of residues. Subtle changes in exchange rates are also visible around the arabinose binding pocket and in the DNA binding domain.
arabinose binding pocketEscherichia coli AraC Transcriptional Regulatorpeptide backbone dihedral anglesrepresses expressionN 168RNA polymerase transcriptionC-terminal enddimerizing α- helixdimerization domains forces AraCDNA half-siteslinker residuesaraBAD catabolism genesexchange ratesdimeric AraC proteinDOIDNA binding domainDNA binding domainsquaternary repositioningSubtle changesinterdomain linkeraraBAD promoterArabinose Altersl-arabinose operondimeric Escherichia coliarabinose-induced destabilizationamide hydrogen-bonded structureAraC conformationdimeric dimerization domainsdimerization domainarabinose allosterically