M. tuberculosis Sliding β-Clamp Does Not Interact Directly with the NAD+ -Dependent DNA Ligase

The sliding β-clamp, an important component of the DNA replication and repair machinery, is drawing increasing attention as a therapeutic target. We report the crystal structure of the M. tuberculosis β-clamp (Mtbβ-clamp) to 3.0 Å resolution. The protein crystallized in the space group C2221 with cell-dimensions a = 72.7, b = 234.9 & c = 125.1 Å respectively. Mtbβ-clamp is a dimer, and exhibits head-to-tail association similar to other bacterial clamps. Each monomer folds into three domains with similar structures respectively and associates with its dimeric partner through 6 salt-bridges and about 21 polar interactions. Affinity experiments involving a blunt DNA duplex, primed-DNA and nicked DNA respectively show that Mtbβ-clamp binds specifically to primed DNA about 1.8 times stronger compared to the other two substrates and with an apparent Kd of 300 nM. In bacteria like E. coli, the β-clamp is known to interact with subunits of the clamp loader, NAD+ -dependent DNA ligase (LigA) and other partners. We tested the interactions of the Mtbβ-clamp with MtbLigA and the γ-clamp loader subunit through radioactive gel shift assays, size exclusion chromatography, yeast-two hybrid experiments and also functionally. Intriguingly while Mtbβ-clamp interacts in vitro with the γ-clamp loader, it does not interact with MtbLigA unlike in bacteria like E. coli where it does. Modeling studies involving earlier peptide complexes reveal that the peptide-binding site is largely conserved despite lower sequence identity between bacterial clamps. Overall the results suggest that other as-yet-unidentified factors may mediate interactions between the clamp, LigA and DNA in mycobacteria

Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining

Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells de-pendent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining inter-molecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based as-say of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding do-main, one of three domains constituting the LigIII cat-alytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging in-termediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation

The mitochondrial single-stranded DNA binding protein from S. cerevisiae, Rim1, does not form stable homo-tetramers and binds DNA as a dimer of dimers

Rim1 is the mitochondrial single-stranded DNA binding protein in Saccharomyces cerevisiae and functions to coordinate replication and maintenance of mtDNA. Rim1 can form homo-tetramers in solution and this species has been assumed to be solely responsible for ssDNA binding. We solved structures of tetrameric Rim1 in two crystals forms which differ in the relative orientation of the dimers within the tetramer. In testing whether the different arrangement of the dimers was due to formation of unstable tetramers, we discovered that while Rim1 forms tetramers at high protein concentration, it dissociates into a smaller oligomeric species at low protein concentrations. A single point mutation at the dimer–dimer interface generates stable dimers and provides support for a dimer–tetramer oligomerization model. The presence of Rim1 dimers in solution becomes evident in DNA binding studies using short ssDNA substrates. However, binding of the first Rim1 dimer is followed by binding of a second dimer, whose affinity depends on the length of the ssDNA. We propose a model where binding of DNA to a dimer of Rim1 induces tetramerization, modulated by the ability of the second dimer to interact with ssDNA

Phosphorylation-Dependent Conformations of the Disordered Carboxyl-Terminus Domain in the Epidermal Growth Factor Receptor

© 2020 American Chemical Society. All rights reserved. The epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, regulates basic cellular functions and is a major target for anticancer therapeutics. The carboxyl-terminus domain is a disordered region of EGFR that contains the tyrosine residues, which undergo autophosphorylation followed by docking of signaling proteins. Local phosphorylation-dependent secondary structure has been identified and is thought to be associated with the signaling cascade. Deciphering and distinguishing the overall conformations, however, have been challenging because of the disordered nature of the carboxyl-terminus domain and resultant lack of well-defined three-dimensional structure for most of the domain. We investigated the overall conformational states of the isolated EGFR carboxyl-terminus domain using single-molecule Förster resonance energy transfer and coarse-grained simulations. Our results suggest that electrostatic interactions between charged residues emerge within the disordered domain upon phosphorylation, producing a looplike conformation. This conformation may enable binding of downstream signaling proteins and potentially reflect a general mechanism in which electrostatics transiently generate functional architectures in disordered regions of a well-folded protein

Interactions of the <i>M.tuberculosis</i> and <i>E. coli</i> β-Clamps with various proteins.

<p>Mtbβ-Clamp^OG (90 nM) was titrated with increasing concentration of the γ-clamp loader (Rv3721c) and LigA respectively (labeled in the figure). Interactions between the β-Clamp and LigA from <i>E. coli</i> was used as positive control where <i>E.coli</i> β-Clamp^OG (90 nM) was titrated with increasing concentration of <i>E.coli</i> LigA, and MtbLigA respectively. The <i>E. coli</i> LigA was also titrated against the Mtbβ-Clamp^OG to probe for their interactions too. BSA was used as a control for non-specific interactions. Changes to the relative fluorescence intensity were observed at λ_max 510.</p

Radioactive Gel shift assay to probe for MtbLigA-Mtbβ-Clamp interactions.

<p>P³² labelled Mtbβ-Clamp (90 nM) was titrated against increasing concentration of MtbLigA. Samples were analysed on 6% Native PAGE. Shifts were analysed by autoradiography. No interaction could be detected between the proteins.</p

Peptide binding groove of the Mtbβ-Clamp, structural alignments and inhibitor interactions.

<p>(<b>A</b>) The hydrophobic groove, depicted in <i>yellow</i>, is present between domains II and III of each protomer. The residues of the respective hydrophobic grooves were found to be quite conserved among bacterial β-clamps. (<b>B</b>) Structural alignment of the peptide binding groove from the respective crystal structures of the Mtbβ-clamp (<i>blue</i>) and <i>E. coli</i> β-clamp (<i>red</i>). The Mtbβ-Clamp residues are numbered. (<b>C</b>) Superposition of the Mtbβ-Clamp crystal structure onto that of the <i>E. coli</i> β-clamp -RU7 inhibitor complex (PDB: 3D1G). The surface of the Mtbβ-clamp is colored <i>green</i> while the peptide binding pocket of Mtbβ-Clamp is colored according to the sequence conservation between Mtbβ-clamp and <i>E. coli</i> β-clamp; <i>dark pink</i> represent identical residues while <i>light pink</i> represents homologous residues. The RU7 inhibitor is depicted in <i>cyan</i> stick representation. The Pol IV peptide (yellow, stick representation) from its crystal structure complex with the <i>E. coli</i> clamp (PDB: 3D1E) is also shown. The inhibitor mainly interacts with subsite 1 of the peptide binding site.</p

Polar inter-subunit interactions in the Mtbβ-Clamp dimer.

<p>Polar inter-subunit interactions in the Mtbβ-Clamp dimer.</p

Far UV CD, relative activity and thermal unfolding of the proteins.

<p>(A) Far UV CD scans between 190–260 nm of the type-1, -type2 and mutant LinA proteins show that the mutants are well-folded comparable to the corresponding native proteins (B) The relative activity (%) is shown plotted against time (min). The mutants correspond to those detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050373#pone-0050373-t003" target="_blank">Table3</a>. Residual α-HCH dehydrochlorinase activity was measured after pre-incubation of LinA proteins at 60°C for different time periods. Activities prior to pre-incubation were taken as 100%. (C) Thermal unfolding of the proteins followed by Circular dichroism spectroscopy. Clearly mutants that contain Q20 and G23 exhibit higher thermostability comparable to that of the native -type2 protein.</p