Allosteric regulation within the highly interconnected structural scaffold of AraC/XylS homologs tolerates a wide range of amino acid changes.

To create bacterial transcription "circuits" for biotechnology, one approach is to recombine natural transcription factors, promoters, and operators. Additional novel functions can be engineered from existing transcription factors such as the E. coli AraC transcriptional activator, for which binding to DNA is modulated by binding L-arabinose. Here, we engineered chimeric AraC/XylS transcription activators that recognized ara DNA binding sites and responded to varied effector ligands. The first step, identifying domain boundaries in the natural homologs, was challenging because (i) no full-length, dimeric structures were available and (ii) extremely low sequence identities (≤10%) among homologs precluded traditional assemblies of sequence alignments. Thus, to identify domains, we built and aligned structural models of the natural proteins. The designed chimeric activators were assessed for function, which was then further improved by random mutagenesis. Several mutational variants were identified for an XylS•AraC chimera that responded to benzoate; two enhanced activation to near that of wild-type AraC. For an RhaR•AraC chimera, a variant with five additional substitutions enabled transcriptional activation in response to rhamnose. These five changes were dispersed across the protein structure, and combinatorial experiments testing subsets of substitutions showed significant non-additivity. Combined, the structure modeling and epistasis suggest that the common AraC/XylS structural scaffold is highly interconnected, with complex intra-protein and inter-domain communication pathways enabling allosteric regulation. At the same time, the observed epistasis and the low sequence identities of the natural homologs suggest that the structural scaffold and function of transcriptional regulation are nevertheless highly accommodating of amino acid changes.

Small-molecule inhibitor of the Shigella flexneri master virulence regulator VirF.

VirF is an AraC family transcriptional activator that is required for the expression of virulence genes associated with invasion and cell-to-cell spread by Shigella flexneri, including multiple components of the type three secretion system (T3SS) machinery and effectors. We tested a small-molecule compound, SE-1 (formerly designated OSSL_051168), which we had identified as an effective inhibitor of the AraC family proteins RhaS and RhaR, for its ability to inhibit VirF. Cell-based reporter gene assays with Escherichia coli and Shigella, as well as in vitro DNA binding assays with purified VirF, demonstrated that SE-1 inhibited DNA binding and transcription activation (likely by blocking DNA binding) by VirF. Analysis of mRNA levels using real-time quantitative reverse transcription-PCR (qRT-PCR) further demonstrated that SE-1 reduced the expression of the VirF-dependent virulence genes icsA, virB, icsB, and ipaB in Shigella. We also performed eukaryotic cell invasion assays and found that SE-1 reduced invasion by Shigella. The effect of SE-1 on invasion required preincubation of Shigella with SE-1, in agreement with the hypothesis that SE-1 inhibited the expression of VirF-activated genes required for the formation of the T3SS apparatus and invasion. We found that the same concentrations of SE-1 had no detectable effects on the growth or metabolism of the bacterial cells or the eukaryotic host cells, respectively, indicating that the inhibition of invasion was not due to general toxicity. Overall, SE-1 appears to inhibit transcription activation by VirF, exhibits selectivity toward AraC family proteins, and has the potential to be developed into a novel antibacterial agent.

The AraC/XylS family activator RhaS negatively autoregulates rhaSR expression by preventing cyclic AMP receptor protein activation.

The Escherichia coli RhaR protein activates expression of the rhaSR operon in the presence of its effector, L-rhamnose. The resulting RhaS protein (plus L-rhamnose) activates expression of the L-rhamnose catabolic and transport operons, rhaBAD and rhaT, respectively. Here, we further investigated our previous finding that rhaS deletion resulted in a threefold increase in rhaSR promoter activity, suggesting RhaS negative autoregulation of rhaSR. We found that RhaS autoregulation required the cyclic AMP receptor protein (CRP) binding site at rhaSR and that RhaS was able to bind to the RhaR binding site at rhaSR. In contrast to the expected repression, we found that in the absence of both RhaR and the CRP binding site at the rhaSR promoter, RhaS activated expression to a level comparable with RhaR activation of the same promoter. However, when the promoter included the RhaR and CRP binding sites, the level of activation by RhaS and CRP was much lower than that by RhaR and CRP, suggesting that CRP could not fully coactivate with RhaS. Taken together, our results indicate that RhaS negative autoregulation involves RhaS competition with RhaR for binding to the RhaR binding site at rhaSR. Although RhaS and RhaR activate rhaSR transcription to similar levels, CRP cannot effectively coactivate with RhaS. Therefore, once RhaS reaches a relatively high protein concentration, presumably sufficient to saturate the RhaS-activated promoters, there will be a decrease in rhaSR transcription. We propose a model in which differential DNA bending by RhaS and RhaR may be the basis for the difference in CRP coactivation.

Differences in the mechanism of the allosteric l-rhamnose responses of the AraC/XylS family transcription activators RhaS and RhaR.

Proteins in the largest subset of AraC/XylS family transcription activators, including RhaS and RhaR, have C-terminal domains (CTDs) that mediate DNA-binding and transcription activation, and N-terminal domains (NTDs) that mediate dimerization and effector binding. The mechanism of the allosteric effector response in this family has been identified only for AraC. Here, we investigated the mechanism by which RhaS and RhaR respond to their effector, l-rhamnose. Unlike AraC, N-terminal truncations suggested that RhaS and RhaR do not use an N-terminal arm to inhibit activity in the absence of effector. We used random mutagenesis to isolate RhaS and RhaR variants with enhanced activation in the absence of l-rhamnose. NTD substitutions largely clustered around the predicted l-rhamnose-binding pockets, suggesting that they mimic the structural outcome of effector binding to the wild-type proteins. RhaS-CTD substitutions clustered in the first HTH motif, and suggested that l-rhamnose induces improved DNA binding. In contrast, RhaR-CTD substitutions clustered at a single residue in the second HTH motif, at a position consistent with improved RNAP contacts. We propose separate allosteric mechanisms for the two proteins: Without l-rhamnose, RhaS does not effectively bind DNA while RhaR does not effectively contact RNAP. Upon l-rhamnose binding, both proteins undergo structural changes that enable transcription activation.

Functional consequences of exchanging domains between LacI and PurR are mediated by the intervening linker sequence.

Homologue function can be differentiated by changing residues that affect binding sites or long-range interactions. LacI and PurR are two proteins that represent the LacI/GalR family (>500 members) of bacterial transcription regulators. All members have distinct DNA-binding and regulatory domains linked by approximately 18 amino acids. Each homologue has specificity for different DNA and regulatory effector ligands; LacI and PurR also exhibit differences in allosteric communication between DNA and effector binding sites. A comparative study of LacI and PurR suggested that alterations in the interface between the regulatory domain and linker are important for differentiating their functions. Four residues (equivalent to LacI positions 48, 55, 58, and 61) appear particularly important for creating a unique interface and were predicted to be necessary for allosteric regulation. However, nearby residues in the linker interact with DNA ligand. Thus, differences observed in interactions between linker and regulatory domain may be the cause of altered function or an effect of the two proteins binding different DNA ligands. To separate these possibilities, we created a chimeric protein with the LacI DNA-binding domain/linker and the PurR regulatory domain (LLhP). If the interface requires homologue-specific interactions in order to propagate the signal from effector binding, then LLhP repression should not be allosterically regulated by effector binding. Experiments show that LLhP is capable of repression from lacO1 and, contrary to expectation, allosteric response is intact. Further, restoring the potential for PurR-like interactions via substitutions in the LLhP linker tends to diminish repression. These effects are especially pronounced for residues 58 and 61. Clearly, binding affinity of LLhP for the lacO1 DNA site is sensitive to long-range changes in the linker. This result also raises the possibility that mutations at positions 58 and 61 co-evolved with changes in the DNA-binding site. In addition, repression measured in the absence and presence of effector ligand shows that allosteric response increases for several LLhP variants with substitutions at positions 48 and 55. Thus, while side chain variation at these sites does not generally dictate the presence or absence of allostery, the nature of the amino acid can modulate the response to effector.

1.65†Šresolution structure of the AraC-family transcriptional activator ToxT from Vibrio cholerae.

ToxT is an AraC-family transcriptional activator protein that controls the expression of key virulence factors in Vibrio cholerae, the causative agent of cholera. ToxT directly activates the expression of the genes that encode the toxin-coregulated pilus and cholera toxin, and also positively auto-regulates its own expression from the tcp promoter. The crystal structure of ToxT has previously been solved at 1.9 Šresolution (PDB entry 3gbg). In this study, a crystal structure of ToxT at 1.65 Šresolution with a similar overall structure to the previously determined structure is reported. However, there are distinct differences between the two structures, particularly in the region that extends from Asp101 to Glu110. This region, which can influence ToxT activity but was disordered in the previous structure, can be traced entirely in the current structure.

3-Substituted Biquinolinium Inhibitors of AraC Family Transcriptional Activator VirF from S. flexneri Obtained Through In Situ Chemical Ionization of 3,4-Disubstituted Dihydroquinolines.

During a structure-activity relationship optimization campaign to develop an inhibitor of AraC family transcriptional activators, we discovered an unexpected transformation of a previously reported inhibitor that occurs under the assay conditions. Once placed in the assay media, the 3, 4-disubstituted dihydroquinoline core of the active analogue rapidly undergoes a decomposition reaction to a quaternary 3-substituted biquinolinium. Further examination established an SAR for this chemotype while also demonstrating its resilience to irreversible binding of biologically relevant nucleophiles.

Identification of a small-molecule inhibitor of bacterial AraC family activators.

Protein members of the AraC family of bacterial transcriptional activators have great promise as targets for the development of novel antibacterial agents. Here, we describe an in vivo high-throughput screen to identify inhibitors of the AraC family activator protein RhaS. The screen used two Escherichia coli reporter fusions: one to identify potential RhaS inhibitors and a second to eliminate nonspecific inhibitors from consideration. One compound with excellent selectivity, OSSL_051168, was chosen for further study. OSSL_051168 inhibited in vivo transcription activation by the RhaS DNA-binding domain to the same extent as the full-length protein, indicating that this domain was the target of its inhibition. Growth curves showed that OSSL_051168 did not affect bacterial cell growth at the concentrations used in this study. In vitro DNA-binding assays with purified protein suggest that OSSL_051168 inhibits DNA binding by RhaS. In addition, we found that it inhibits DNA binding by a second AraC family protein, RhaR, which shares 30% amino acid identity with RhaS. OSSL_051168 did not have a significant impact on DNA binding by the non-AraC family proteins CRP and LacI, suggesting that the inhibition is likely specific for RhaS, RhaR, and possibly additional AraC family activator proteins.

Linker regions of the RhaS and RhaR proteins.

Substitutions within the interdomain linkers of the AraC/XylS family proteins RhaS and RhaR were tested to determine whether side chain identity or linker structure was required for function. Neither was found crucial, suggesting that the linkers do not play a direct role in activation, but rather simply connect the two domains.

Transcription activation by the DNA-binding domain of the AraC family protein RhaS in the absence of its effector-binding domain.

The Escherichia coli L-rhamnose-responsive transcription activators RhaS and RhaR both consist of two domains, a C-terminal DNA-binding domain and an N-terminal dimerization domain. Both function as dimers and only activate transcription in the presence of L-rhamnose. Here, we examined the ability of the DNA-binding domains of RhaS (RhaS-CTD) and RhaR (RhaR-CTD) to bind to DNA and activate transcription. RhaS-CTD and RhaR-CTD were both shown by DNase I footprinting to be capable of binding specifically to the appropriate DNA sites. In vivo as well as in vitro transcription assays showed that RhaS-CTD could activate transcription to high levels, whereas RhaR-CTD was capable of only very low levels of transcription activation. As expected, RhaS-CTD did not require the presence of L-rhamnose to activate transcription. The upstream half-site at rhaBAD and the downstream half-site at rhaT were found to be the strongest of the known RhaS half-sites, and a new putative RhaS half-site with comparable strength to known sites was identified. Given that cyclic AMP receptor protein (CRP), the second activator required for full rhaBAD expression, cannot activate rhaBAD expression in a DeltarhaS strain, it was of interest to test whether CRP could activate transcription in combination with RhaS-CTD. We found that RhaS-CTD allowed significant activation by CRP, both in vivo and in vitro, although full-length RhaS allowed somewhat greater CRP activation. We conclude that RhaS-CTD contains all of the determinants necessary for transcription activation by RhaS.

Cyclic AMP receptor protein and RhaR synergistically activate transcription from the L-rhamnose-responsive rhaSR promoter in Escherichia coli.

The Escherichia coli rhaSR operon encodes two AraC family transcription activator proteins, RhaS and RhaR, which regulate expression of the l-rhamnose catabolic regulon in response to l-rhamnose availability. RhaR positively regulates rhaSR in response to l-rhamnose, and RhaR activation can be enhanced by the cyclic AMP (cAMP) receptor protein (CRP) protein. CRP is a well-studied global transcription regulator that binds to DNA as a dimer and activates transcription in the presence of cAMP. We investigated the mechanism of CRP activation at rhaSR both alone and in combination with RhaR in vivo and in vitro. Base pair substitutions at potential CRP binding sites in the rhaSR-rhaBAD intergenic region demonstrate that CRP site 3, centered at position -111.5 relative to the rhaSR transcription start site, is required for the majority of the CRP-dependent activation of rhaSR. DNase I footprinting confirms that CRP binds to site 3; CRP binding to the other potential CRP sites at rhaSR was not detected. We show that, at least in vitro, CRP is capable of both RhaR-dependent and RhaR-independent activation of rhaSR from a total of three transcription start sites. In vitro transcription assays indicate that the carboxy-terminal domain of the alpha subunit (alpha-CTD) of RNA polymerase is at least partially dispensable for RhaR-dependent activation but that the alpha-CTD is required for CRP activation of rhaSR. Although CRP requires the presence of RhaR for efficient in vivo activation of rhaSR, DNase I footprinting assays indicated that cooperative binding between RhaR and CRP does not make a significant contribution to the mechanism of CRP activation at rhaSR. It therefore appears that CRP activates transcription from rhaSR as it would at simple class I promoters, albeit from a relatively distant position.

Amino acid contacts between sigma 70 domain 4 and the transcription activators RhaS and RhaR.

The RhaS and RhaR proteins are transcription activators that respond to the availability of L-rhamnose and activate transcription of the operons in the Escherichia coli L-rhamnose catabolic regulon. RhaR activates transcription of rhaSR, and RhaS activates transcription of the operon that encodes the L-rhamnose catabolic enzymes, rhaBAD, as well as the operon that encodes the L-rhamnose transport protein, rhaT. RhaS is 30% identical to RhaR at the amino acid level, and both are members of the AraC/XylS family of transcription activators. The RhaS and RhaR binding sites overlap the -35 hexamers of the promoters they regulate, suggesting they may contact the sigma70 subunit of RNA polymerase as part of their mechanisms of transcription activation. In support of this hypothesis, our lab previously identified an interaction between RhaS residue D241 and sigma70 residue R599. In the present study, we first identified two positively charged amino acids in sigma70, K593 and R599, and three negatively charged amino acids in RhaR, D276, E284, and D285, that were important for RhaR-mediated transcription activation of the rhaSR operon. Using a genetic loss-of-contact approach we have obtained evidence for a specific contact between RhaR D276 and sigma70 R599. Finally, previous results from our lab separately showed that RhaS D250A and sigma70 K593A were defective at the rhaBAD promoter. Our genetic loss-of-contact analysis of these residues indicates that they identify a second site of contact between RhaS and sigma70.