Different Behaviors In Vivo of Mutations in the β Hairpin Loop of the DNA Polymerases of the Closely Related Phages T4 and RB69

The T4 and RB69 DNA replicative polymerases are members of the B family and are highly similar. Both replicate DNA with high fidelity and employ the same mechanism that allows efficient switching of the primer terminus between the polymerase and exonuclease sites. Both polymerases have a β hairpin loop (hereafter called the β loop) in their exonuclease domains that plays an important role in active-site switching. The β loop is involved in strand separation and is needed to stabilize partially strand-separated exonuclease complexes. In T4 DNA polymerase, modification of the β-loop residue G255 to Ser confers a strong mutator phenotype in vivo due to a reduced ability to form editing complexes. Here, we describe the RB69 DNA polymerase mutant with the equivalent residue (G258) changed to Ser but showing only mild mutator activity in vivo. On the other hand, deletion of the tip of the RB69 β loop confers a strong mutator phenotype in vivo. Based on detailed mutational spectral analyses, DNA binding activities, and coupled polymerase/exonuclease assays, we define the differences between the T4 and RB69 polymerases. We propose that their β loops facilitate strand separation in both polymerases, while the residues that form the loop have low structural constraints.     

Structural and thermodynamic signatures of DNA recognition by Mycobacterium tuberculosis DnaA

An essential protein, DnaA, binds to 9-bp DNA sites within the origin of replication oriC. These binding events are prerequisite to forming an enigmatic nucleoprotein scaffold that initiates replication. The number, sequences, positions, and orientations of these short DNA sites, or DnaA boxes, within the oriCs of different bacteria vary considerably. To investigate features of DnaA boxes that are important for binding Mycobacterium tuberculosis DnaA (MtDnaA), we have determined the crystal structures of the DNA binding domain (DBD) of MtDnaA bound to a cognate MtDnaA-box (at 2.0 Å resolution) and to a consensus Escherichia coli DnaA-box (at 2.3 Å). These structures, complemented by calorimetric equilibrium binding studies of MtDnaA DBD in a series of DnaA-box variants, reveal the main determinants of DNA recognition and establish the [T/C][T/A][G/A]TCCACA sequence as a high-affinity MtDnaA-box. Bioinformatic and calorimetric analyses indicate that DnaA-box sequences in mycobacterial oriCs generally differ from the optimal binding sequence. This sequence variation occurs commonly at the first 2 bp, making an in vivo mycobacterial DnaA-box effectively a 7-mer and not a 9-mer. We demonstrate that the decrease in the affinity of these MtDnaA-box variants for MtDnaA DBD relative to that of the highest-affinity box TTGTCCACA is less than 10-fold. The understanding of DnaA-box recognition by MtDnaA and E. coli DnaA enables one to map DnaA-box sequences in the genomes of M. tuberculosis and other eubacteria.     

Mechanism for Attenuation of DNA Binding by MarR Family Transcriptional Regulators by Small Molecule Ligands

Members of the multiple antibiotic resistance regulator (MarR) family control gene expression in a variety of metabolic processes in bacteria and archaea. Hypothetical uricase regulator (HucR), which belongs to the ligand-responsive branch of the MarR family, regulates uricase expression in Deinococcus radiodurans by binding a shared promoter region between uricase and HucR genes. We show here that HucR responds only to urate and, to a lesser extent, to xanthine by attenuated DNA binding, compared to other intermediates of purine degradation. Using molecular-dynamics-guided mutational analysis, we identified the ligand-binding site in HucR. Electrophoretic mobility shift assays and intrinsic Trp fluorescence have identified W20 from the N-terminal helix and R80 from helix 3, which serves as a scaffold for the DNA recognition helix, as being essential for ligand binding. Using structural data combined with in silico and in vitro analyses, we propose a mechanism for the attenuation of DNA binding in which a conformational change initiated by charge repulsion due to a bound ligand propagates to DNA recognition helices. This mechanism may apply generally to MarR homologs that bind anionic phenolic ligands.     

Structural basis for promoter DNA recognition by the response regulator OmpR

OmpR, a response regulator of the EnvZ/OmpR two-component system (TCS), controls the reciprocal regulation of two porin proteins, OmpF and OmpC, in bacteria. During signal transduction, OmpR (OmpR-FL) undergoes phosphorylation at its conserved Asp residue in the N-terminal receiver domain (OmpRn) and recognizes the promoter DNA from its C-terminal DNA-binding domain (OmpRc) to elicit an adaptive response. Apart from that, OmpR regulates many genes in Escherichia coli and is important for virulence in several pathogens. However, the molecular mechanism of the regulation and the structural basis of OmpR–DNA binding is still not fully clear. In this study, we presented the crystal structure of OmpRc in complex with the F1 region of the ompF promoter DNA from E. coli. Our structural analysis suggested that OmpRc binds to its cognate DNA as a homodimer, only in a head-to-tail orientation. Also, the OmpRc apo-form showed a unique domain-swapped crystal structure under different crystallization conditions. Biophysical experimental data, such as NMR, fluorescent polarization and thermal stability, showed that inactive OmpR-FL (unphosphorylated) could bind to promoter DNA with a weaker binding affinity as compared with active OmpR-FL (phosphorylated) or OmpRc, and also confirmed that phosphorylation may only enhance DNA binding. Furthermore, the dimerization interfaces in the OmpRc–DNA complex structure identified in this study provide an opportunity to understand the regulatory role of OmpR and explore the potential for this “druggable” target.     

Binding of the Lactococcal Drug Dependent Transcriptional Regulator LmrR to Its Ligands and Responsive Promoter Regions

The heterodimeric ABC transporter LmrCD from Lactococcus lactis is able to extrude several different toxic compounds from the cell, fulfilling a role in the intrinsic and induced drug resistance. The expression of the lmrCD genes is regulated by the multi-drug binding repressor LmrR, which also binds to its own promoter to autoregulate its own expression. Previously, we reported the crystal structure of LmrR in the presence and absence of the drugs Hoechst 33342 and daunomycin. Analysis of the mechanism how drugs control the repressor activity of LmrR is impeded by the fact that these drugs also bind to DNA. Here we identified, using X-ray crystallography and fluorescence, that riboflavin binds into the drug binding cavity of LmrR, adopting a similar binding mode as Hoechst 33342 and daunomycin. Microscale thermophoresis was employed to quantify the binding affinity of LmrR to its responsive promoter regions and to evaluate the cognate site of LmrR in the lmrCD promoter region. Riboflavin reduces the binding affinity of LmrR for the promoter regions. Our results support a model wherein drug binding to LmrR relieves the LmrR dependent repression of the lmrCD genes.     

Pattern recognition with a fibril-specific antibody fragment reveals the surface variability of natural amyloid fibrils

Amyloid immunotherapy has led to the rise of antibodies, which target amyloid fibrils or structural precursors of fibrils, based on their specific conformational properties. Recently, we reported the biotechnological generation of the B10 antibody fragment, which provides conformation-specific binding to amyloid fibrils. B10 strongly interacts with fibrils from Alzheimer's β amyloid (Aβ) peptide, while disaggregated Aβ peptide or Aβ oligomers are not explicitly recognized. B10 also enables poly-amyloid-specific binding and recognizes amyloid fibrils derived from different types of amyloidosis or different polypeptide chains. Based on our current data, however, we find that B10 does not recognize all tested amyloid fibrils and amyloid tissue deposits. It also does not specifically interact with intrinsically unfolded polypeptide chains or globular proteins even if the latter encompass high β-sheet content or β-solenoid domains. By contrast, B10 binds amyloid fibrils from d-amino acid or l-amino acid peptides and non-proteinaceous biopolymers with highly regular and anionic surface properties, such as heparin and DNA. These data establish that B10 binding does not depend on an amyloid-specific or protein-specific backbone structure. Instead, it involves the recognition of a highly regular and anionic surface pattern. This specificity mechanism is conserved in nature and occurs also within a group of natural amyloid receptors from the innate immune system, the pattern recognition receptors. Our data illuminate the structural diversity of naturally occurring amyloid scaffolds and enable the discrimination of distinct fibril populations in vitro and within diseased tissues.     

Functional characterization of the Escherichia coli Fis-DNA binding sequence

The Escherichia coli protein Fis is remarkable for its ability to interact specifically with DNA sites of highly variable sequences. The mechanism of this sequence-flexible DNA recognition is not well understood. In a previous study, we examined the contributions of Fis residues to high-affinity binding at different DNA sequences using alanine-scanning mutagenesis and identified several key residues for Fis-DNA recognition. In this work, we investigated the contributions of the 15-bp core Fis binding sequence and its flanking regions to Fis-DNA interactions. Systematic base-pair replacements made in both half sites of a palindromic Fis binding sequence were examined for their effects on the relative Fis binding affinity. Missing contact assays were also used to examine the effects of base removal within the core binding site and its flanking regions on the Fis-DNA binding affinity. The results revealed that: (1) the − 7G and + 3Y bases in both DNA strands (relative to the central position of the core binding site) are major determinants for high-affinity binding; (2) the C⁵ methyl group of thymine, when present at the + 4 position, strongly hinders Fis binding; and (3) AT-rich sequences in the central and flanking DNA regions facilitate Fis-DNA interactions by altering the DNA structure and by increasing the local DNA flexibility. We infer that the degeneracy of specific Fis binding sites results from the numerous base-pair combinations that are possible at noncritical DNA positions (from − 6 to − 4, from − 2 to + 2, and from + 4 to + 6), with only moderate penalties on the binding affinity, the roughly similar contributions of − 3A or G and + 3T or C to the binding affinity, and the minimal requirement of three of the four critical base pairs to achieve considerably high binding affinities.     

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.