Decreased Sensitivity to Changes in the Concentration of Metal Ions as the Basis for the Hyperactivity of DtxR(E175K)

The metal-ion-activated diphtheria toxin repressor (DtxR) is responsible for the regulation of virulence and other genes in Corynebacterium diphtheriae. A single point mutation in DtxR, DtxR(E175K), causes this mutant repressor to have a hyperactive phenotype. Mice infected with Mycobacterium tuberculosis transformed with plasmids carrying this mutant gene show reduced signs of the tuberculosis infection. Corynebacterial DtxR is able to complement mycobacterial IdeR and vice versa. To date, an explanation for the hyperactivity of DtxR(E175K) has remained elusive. In an attempt to address this issue, we have solved the first crystal structure of DtxR(E175K) and characterized this mutant using circular dichroism, isothermal titration calorimetry, and other biochemical techniques. The results show that although DtxR(E175K) and the wild type have similar secondary structures, DtxR(E175K) gains additional thermostability upon activation with metal ions, which may lead to this mutant requiring a lower concentration of metal ions to reach the same levels of thermostability as the wild-type protein. The E175K mutation causes binding site 1 to retain metal ion bound at all times, which can only be removed by incubation with an ion chelator. The crystal structure of DtxR(E175K) shows an empty binding site 2 without evidence of oxidation of Cys102. The association constant for this low-affinity binding site of DtxR(E175K) obtained from calorimetric titration with Ni(II) is K_a = 7.6 ± 0.5 × 10⁴, which is very similar to the reported value for the wild-type repressor, K_a = 6.3 × 10⁴. Both the wild type and DtxR(E175K) require the same amount of metal ion to produce a shift in the electrophoretic mobility shift assay, but unlike the wild type, DtxR(E175K) binding to its cognate DNA [tox promoter-operator (toxPO)] does not require metal-ion supplementation in the running buffer. In the timescale of these experiments, the Mn(II)-DtxR(E175K)-toxPO complex is insensitive to changes in the environmental cation concentrations. In addition to Mn(II), Ni(II), Co(II), Cd(II), and Zn(II) are able to sustain the hyperactive phenotype. These results demonstrate a prominent role of binding site 1 in the activation of DtxR and support the hypothesis that DtxR(E175K) attenuates the expression of virulence due to the decreased ability of the Me(II)-DtxR(E175K)-toxPO complex to dissociate at low concentrations of metal ions.     

Binding the Mammalian High Mobility Group Protein AT-hook 2 to AT-Rich Deoxyoligonucleotides: Enthalpy-Entropy Compensation

HMGA2 is a DNA minor-groove binding protein. We previously demonstrated that HMGA2 binds to AT-rich DNA with very high binding affinity where the binding of HMGA2 to poly(dA-dT)₂ is enthalpy-driven and to poly(dA)poly(dT) is entropy-driven. This is a typical example of enthalpy-entropy compensation. To further study enthalpy-entropy compensation of HMGA2, we used isothermal-titration-calorimetry to examine the interactions of HMGA2 with two AT-rich DNA hairpins: 5′-CCAAAAAAAAAAAAAAAGCCCCCGCTTTTTTTTTTTTTTTGG-3′ (FL-AT-1) and 5′-CCATATATATATATATAGCCCCCGCTATATATATATATATGG-3′ (FL-AT-2). Surprisingly, we observed an atypical isothermal-titration-calorimetry-binding curve at low-salt aqueous solutions whereby the apparent binding-enthalpy decreased dramatically as the titration approached the end. This unusual behavior can be attributed to the DNA-annealing coupled to the ligand DNA-binding and is eliminated by increasing the salt concentration to ∼200 mM. At this condition, HMGA2 binding to FL-AT-1 is entropy-driven and to FL-AT-2 is enthalpy-driven. Interestingly, the DNA-binding free energies for HMGA2 binding to both hairpins are almost temperature independent; however, the enthalpy-entropy changes are dependent on temperature, which is another aspect of enthalpy-entropy compensation. The heat capacity change for HMGA2 binding to FL-AT-1 and FL-AT-2 are almost identical, indicating that the solvent displacement and charge-charge interaction in the coupled folding/binding processes for both binding reactions are similar.     

Bacterial Genome Partitioning: N-Terminal Domain of IncC Protein Encoded by Broad-Host-Range Plasmid RK2 Modulates Oligomerisation and DNA Binding

ParA Walker ATPases form part of the machinery that promotes better-than-random segregation of bacterial genomes. ParA proteins normally occur in one of two forms, differing by their N-terminal domain (NTD) of approximately 100 aa, which is generally associated with site-specific DNA binding. Unusually, and for as yet unknown reasons, parA (incC) of IncP-1 plasmids is translated from alternative start codons producing two forms, IncC1 (364 aa) and IncC2 (259 aa), whose ratio varies between hosts. IncC2 could be detected as an oligomeric form containing dimers, tetramers and octamers, but the N-terminal extension present in IncC1 favours nucleotide-stimulated dimerisation as well as high-affinity and ATP-dependent non-specific DNA binding. The IncC1 NTD does not dimerise or bind DNA alone, but it does bind IncC2 in the presence of nucleotides. Mixing IncC1 and IncC2 improved polymerisation and DNA binding. Thus, the NTD may modulate the polymerisation interface, facilitating polymerisation/depolymerisation and DNA binding, to promote the cycle that drives partitioning.     

A Single-Amino-Acid Substitution in the C Terminus of PhoP Determines DNA-Binding Specificity of the Virulence-Associated Response Regulator from Mycobacterium tuberculosis

The Mycobacterium tuberculosis PhoP-PhoR two-component system is essential for virulence in animal models of tuberculosis. Genetic and biochemical studies indicate that PhoP regulates the expression of more than 110 genes in M. tuberculosis. The C-terminal effector domain of PhoP exhibits a winged helix-turn-helix motif with the molecular surfaces around the recognition helix (α8) displaying strong positive electrostatic potential, suggesting its role in DNA binding and nucleotide sequence recognition. Here, the relative importance of interfacial α8-DNA contacts has been tested through rational mutagenesis coupled with in vitro binding-affinity studies. Most PhoP mutants, each with a potential DNA contacting residue replaced with Ala, had significantly reduced DNA binding affinity. However, substitution of nonconserved Glu215 had a major effect on the specificity of recognition. Although lack of specificity does not necessarily correlate with gross change in the overall DNA binding properties of PhoP, structural superposition of the PhoP C-domain on the Escherichia coli PhoB C-domain-DNA complex suggests a base-specific interaction between Glu215 of PhoP and the ninth base of the DR1 repeat motif. Biochemical experiments corroborate these results, showing that DNA recognition specificity can be altered by as little as a single residue change of the protein or a single base change of the DNA. The results have implications for the mechanism of sequence-specific DNA binding by PhoP.     

Assembling the human IFN-beta enhanceosome in solution

Assembly of interferon-β enhanceosome from its individual protein components and of enhancer DNA has been studied in solution using a combination of fluorescence anisotropy, microcalorimetry, and CD titration. It was shown that the enhancer binds only one full-length phosphomimetic IRF-3 dimer at the PRDIII-PRDI sites, and this binding does not exhibit cooperativity with binding of the ATF-2/c-Jun bZIP (leucine zipper dimer with basic DNA recognition segments) heterodimer at the PRDIV site. The orientation of the bZIP pair is, therefore, not determined by the presence of the IRF-3 dimer, but is predetermined by the asymmetry of the PRDIV site. In contrast, bound IRF-3 dimer interacts strongly with the NF-κB (p50/p65) heterodimer bound at the neighboring PRDII site. The orientation of bound NF-κB is also predetermined by the asymmetry of the PRDII site and is the opposite of that found in the crystal structure. The HMG-I/Y protein, proposed as orchestrating enhanceosome assembly, interacts specifically with the PRDII site of the interferon-β enhancer by inserting its DNA-binding segments (AT hooks) into the minor groove, resulting in a significant increase in NF-κB binding affinity for the major groove of this site.     

DNA binding mode transitions of Escherichia coli HU(alphabeta): evidence for formation of a bent DNA--protein complex on intact, linear duplex DNA

Escherichia coli HU_αβ, a major nucleoid-associated protein, organizes chromosomal DNA and facilitates numerous DNA transactions. Using isothermal titration calorimetry, fluorescence resonance energy transfer and a series of DNA lengths (8 bp, 15 bp, 34 bp, 38 bp and 160 bp) we established that HU_αβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA molar ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 bp), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 ± 0.4 × 10⁶ M^− 1, and a binding enthalpy of + 7.7 ± 0.6 kcal/mol at 15 °C and 0.15 M Na⁺. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω∼20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 ± 0.2 × 10⁵ M^− 1 for the 10 bp mode, 3.5 ± 1.4 × 10⁴ M^− 1 for the 6 bp mode) and binding enthalpies (4.2 ± 0.3 kcal/mol for the 10 bp mode, − 1.6 ± 0.3 kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. Fluorescence resonance energy transfer data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 bp and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement), fluorescence, and electrophoretic gel mobility-shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA.     

TolA Modulates the Oligomeric Status of YbgF in the Bacterial Periplasm

The trans-envelope Tol complex of Gram-negative bacteria is recruited to the septation apparatus during cell division where it is involved in stabilizing the outer membrane. The last gene in the tol operon, ybgF, is highly conserved, yet does not seem to be required for Tol function. We have addressed this anomaly by characterizing YbgF from Escherichia coli and its interaction with TolA, which, based on previous yeast two-hybrid data, is the only known physical link between YbgF and the Tol system. We show that the stable YbgF trimer undergoes a marked change in oligomeric state on binding TolA, forming a one-to-one complex with the Tol protein. Through a combination of pull-down assays, deletion analysis, and isothermal titration calorimetry, we map the TolA-YbgF interface to the C-terminal tetratricopeptide repeat domain of YbgF and 31 residues at the C-terminal end of TolA domain II (TolA^280-313). We show that TolB, which binds TolA domain III close to the YbgF binding site, has no impact on the YbgF-TolA association. We also report the crystal structures of the two component domains of YbgF, the N-terminal coiled coil from E. coli YbgF, which forms a stable trimer and controls the oligomeric status of YbgF, and the monomeric tetratricopeptide repeat domain from Xanthomonas campestris YbgF, which is also able to trimerize. Although the coiled coil is not directly involved in TolA binding, we demonstrate that the regular hydrophilic patterning of its otherwise hydrophobic core is a prerequisite for the TolA-induced oligomeric-state transition of YbgF. We postulate that rather than YbgF affecting Tol function, it is the change in YbgF oligomeric status (with an accompanying change in its function) that likely explains the necessity for tight co-regulation of the ybgF and tol genes in Gram-negative bacteria.     

The Structure of the Biofilm-controlling Response Regulator BfmR from Acinetobacter baumannii Reveals Details of Its DNA-binding Mechanism

The rise of drug-resistant bacterial infections coupled with decreasing antibiotic efficacy poses a significant challenge to global health care. Acinetobacter baumannii is an insidious, emerging bacterial pathogen responsible for severe nosocomial infections aided by its ability to form biofilms. The response regulator BfmR, from the BfmR/S two-component system, is the master regulator of biofilm initiation in A. baumannii and is a tractable therapeutic target. Here we present the structure of A. baumannii BfmR using a hybrid approach combining X-ray crystallography, nuclear magnetic resonance spectroscopy, chemical crosslinking mass spectrometry, and molecular modeling. We also show that BfmR binds the previously proposed bfmRS promoter sequence with moderate affinity. While BfmR shares many traits with other OmpR/PhoB family response regulators, some unusual properties were observed.Most importantly, we observe that when phosphorylated, BfmR binds this promoter sequence with a lower affinity than when not phosphorylated. All other OmpR/PhoB family members studied to date show an increase in DNA-binding affinity upon phosphorylation. Understanding the structural and biochemical mechanisms of BfmR will aid in the development of new antimicrobial therapies.     

Characterization of the functional domains of Escherichia coli RNase II

RNase II is a single-stranded-specific 3'-exoribonuclease that degrades RNA generating 5'-mononucleotides. This enzyme is the prototype of an ubiquitous family of enzymes that are crucial in RNA metabolism and share a similar domain organization. By sequence prediction, three different domains have been assigned to the Escherichia coli RNase II: two RNA-binding domains at each end of the protein (CSD and S1), and a central RNB catalytic domain. In this work we have performed a functional characterization of these domains in order to address their role in the activity of RNase II. We have constructed a large set of RNase II truncated proteins and compared them to the wild-type regarding their exoribonucleolytic activity and RNA-binding ability. The dissociation constants were determined using different single- or double-stranded substrates. The results obtained revealed that S1 is the most important domain in the establishment of stable RNA-protein complexes, and its elimination results in a drastic reduction on RNA-binding ability. In addition, we also demonstrate that the N-terminal CSD plays a very specific role in RNase II, preventing a tight binding of the enzyme to single-stranded poly(A) chains. Moreover, the biochemical results obtained with RNB mutant that lacks both putative RNA-binding domains, revealed the presence of an additional region involved in RNA binding. Such region, was identified by sequence analysis and secondary structure prediction as a third putative RNA-binding domain located at the N-terminal part of RNB catalytic domain.