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. 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.

A genetic and physical study of the interdomain linker of E. Coli AraC protein--a trans-subunit communication pathway.

Genetic experiments with full length AraC and biophysical experiments with its dimerization domain plus linker suggest that arabinose binding to the dimerization domain changes the properties of the inter-domain linker which connects the dimerization domain to the DNA binding domain via interactions that do not depend on the DNA binding domain. Normal AraC function was found to tolerate considerable linker sequence alteration excepting proline substitutions. The proline substitutions partially activate transcription even in the absence of arabinose and hint that a structural shift between helix and coil may be involved. To permit fluorescence anisotropy measurements that could detect arabinose-dependent dynamic differences in the linkers, IAEDANS was conjugated to a cysteine residue substituted at the end of the linker of dimerization domain. Arabinose, but not other sugars, decreased the steady-state anisotropy, indicating either an increase in mobility and/or an increase in the fluorescence lifetime of the IAEDANS. Time-resolved fluorescence measurements showed that the arabinose-induced anisotropy decrease did not result from an increase in the excited-state lifetime. Hence arabinose-induced decreases in anisotropy appear to result from increased tumbling of the fluorophore. Arabinose did not decrease the anisotropy in mutants incapable of binding arabinose nor did it alter the anisotropy when IAEDANS was conjugated elsewhere in the dimerization domain. Experiments with heterodimers of the dimerization domain showed that the binding of arabinose to one subunit of the dimer decreases the fluorescence anisotropy of only a fluorophore on the linker of the other subunit.

Modulation of DNA binding by gene-specific transcription factors.

The transcription of many genes, particularly in prokaryotes, is controlled by transcription factors whose activity can be modulated by controlling their DNA binding affinity. Understanding the molecular mechanisms by which DNA binding affinity is regulated is important, but because forming definitive conclusions usually requires detailed structural information in combination with data from extensive biophysical, biochemical, and sometimes genetic experiments, little is truly understood about this topic. This review describes the biological requirements placed upon DNA binding transcription factors and their consequent properties, particularly the ways that DNA binding affinity can be modulated and methods for its study. What is known and not known about the mechanisms modulating the DNA binding affinity of a number of prokaryotic transcription factors, including CAP and lac repressor, is provided.

Structure and properties of a truely apo form of AraC dimerization domain.

The arabinose-binding pockets of wild type AraC dimerization domains crystallized in the absence of arabinose are occupied with the side chains of Y31 from neighboring domains. This interaction leads to aggregation at high solution concentrations and prevents determination of the structure of truely apo AraC. In this work we found that the aggregation does not significantly occur at physiological concentrations of AraC. We also found that the Y31V mutation eliminates the self-association, but does not affect regulation properties of the protein. At the same time, the mutation allows crystallization of the dimerization domain of the protein with only solvent in the arabinose-binding pocket. Using a distance difference method suitable for detecting and displaying even minor structural variation among large groups of similar structures, we find that there is no significant structural change in the core of monomers of the AraC dimerization domain resulting from arabinose, fucose, or tyrosine occupancy of the ligand-binding pocket. A slight change is observed in the relative orientation of monomers in the dimeric form of the domain upon the binding of arabinose but its significance cannot yet be assessed.

Specific interactions by the N-terminal arm inhibit self-association of the AraC dimerization domain.

Deletion of the regulatory N-terminal arms of the AraC protein from its dimerization domain fragments increases the susceptibility of the dimerization domain to form a series of higher order polymers by indefinite self-association. We investigated how the normal presence of the arm inhibits this self-association. One possibility is that arms can act as an entropic bristles to interfere with the approach of other macromolecules, thereby decreasing collision frequencies. We examined the repulsive effect of flexible arms by measuring the rate of trypsin cleavage of a specially constructed ubiquitin-arm protein. Adding an arm to ubiquitin or increasing its length produced only a modest repulsive effect. This suggests that arms such as the N-terminal arm of AraC do not reduce self-association by entropic exclusion. We consequently tested the hypothesis that the arm on AraC reduces self-association by binding to the core of the dimerization domain even in the absence of arabinose. The behaviors of dimerization domain mutants containing deletions or alterations in the N-terminal arms substantiate this hypothesis. Apparently, interactions between the N-terminal arm and the dimerization domain core position the arm to interfere with the protein-protein contacts necessary for self-association.

A DNA-assisted binding assay for weak protein-protein interactions.

We describe a new method used for quantitating weak interactions between proteins in which the weak interaction is "assisted" by a known DNA-DNA interaction. Oligonucleotides, which are conjugated to proteins of interest, contain short complementary DNA sequences that provide additional binding energy for protein-protein interactions. A stretch of unpaired bases links the protein to the hybridizing DNA sequence to allow formation of both protein-protein and DNA-DNA interactions with minimal structural interference. We validated the DNA-assisted binding method using heterodimerizing coiled-coil proteins. The method was then used to measure the predicted weak interaction between two domains of the Escherichia coli L-arabinose operon regulatory protein AraC. The interaction between domains has the expected magnitude (K(d)=0.37 mM) in the absence of arabinose. Upon addition of arabinose, we detected a weaker and unexpected interaction, which may necessitate modification of the proposed mechanism of AraC. The DNA-assisted binding method may also prove useful in the study of other weak protein-protein interactions.

The salt dependence of the interferon regulatory factor 1 DNA binding domain binding to DNA reveals ions are localized around protein and DNA.

The equilibrium dissociation constant of the DNA binding domain of interferon regulatory factor 1 (IRF1 DBD) for its DNA binding site depends strongly on salt concentration and salt type. These dependencies are consistent with IRF1 DBD binding to DNA, resulting in the release of cations from the DNA and both release of anions from the protein and uptake of a cation by the protein. We demonstrated this by utilizing the fact that the release of fluoride from protein upon complex formation does not contribute to the salt concentration dependence of binding and by studying mutants in which charged residues in IRF1 DBD that form salt bridges with DNA phosphates are changed to alanine. The salt concentration dependencies of the dissociation constants of wild-type IRF1 DBD and the mutants R64A, D73A, K75A, and D73A/K75A were measured in buffer containing NaF, NaCl, or NaBr. The salt concentration and type dependencies of the mutants relative to wild-type IRF1 DBD provide evidence of charge neutralization by solution ions for R64 and by a salt bridge between D73 and K75 in buffer containing chloride or bromide salts. These data also allowed us to determine the number, type, and localization of condensed ions around both IRF1 DBD and its DNA binding site.