Arabinose Alters Both Local and Distal H–D
Exchange Rates in the <i>Escherichia coli</i> AraC Transcriptional
Regulator
Alexander Tischer
Matthew J. Brown
Robert F. Schleif
Matthew Auton
10.1021/acs.biochem.9b00389.s001
https://acs.figshare.com/articles/journal_contribution/Arabinose_Alters_Both_Local_and_Distal_H_D_Exchange_Rates_in_the_i_Escherichia_coli_i_AraC_Transcriptional_Regulator/8298122
In
the absence of arabinose, the dimeric <i>Escherichia coli</i> 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 <i>araBAD</i> 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) <i>Biochemistry</i>, 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 (I<sub>167</sub> and
N<sub>168</sub>). 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 <i>araBAD</i> 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.
2019-06-14 00:00:00
arabinose binding pocket
Escherichia coli AraC Transcriptional Regulator
peptide backbone dihedral angles
represses expression
N 168
RNA polymerase transcription
C-terminal end
dimerizing α- helix
dimerization domains forces AraC
DNA half-sites
linker residues
araBAD catabolism genes
exchange rates
dimeric AraC protein
DOI
DNA binding domain
DNA binding domains
quaternary repositioning
Subtle changes
interdomain linker
araBAD promoter
Arabinose Alters
l-arabinose operon
dimeric Escherichia coli
arabinose-induced destabilization
amide hydrogen-bonded structure
AraC conformation
dimeric dimerization domains
dimerization domain
arabinose allosterically