Interrogating fragments using a protein thermal shift assay

Protein thermal shift is a relatively rapid and inexpensive technique for the identification of low molecular weight compound interactions with protein targets. An increase in the melting temperature of the target protein in the presence of a test ligand is indicative of a promising ligand-protein interaction. Due to its simplicity, protein thermal shift is an attractive method for screening libraries and validating hits in drug discovery programs. The methodology has been used successfully in high throughput screens of small molecule libraries, and its application has been extended to report on protein-drug-like-fragment interactions. Here, we review how protein thermal shift has been employed recently in fragment-based drug discovery (FBDD) efforts, and highlight its application to protein-protein interaction targets. Multiple validation of fragment hits by independent means is paramount to ensure efficient and economical progress in a FBDD campaign. We discuss the applicability of thermal shift assays in this light, and discuss more generally what one does when orthogonal approaches disagree

Disarming Burkholderia pseudomallei: structural and functional characterization of a disulfide oxidoreductase (DsbA) required for virulence in vivo

Aims: The intracellular pathogen Burkholderia pseudomallei causes the disease melioidosis, a major source of morbidity and mortality in southeast Asia and northern Australia. The need to develop novel antimicrobials is compounded by the absence of a licensed vaccine and the bacterium's resistance to multiple antibiotics. In a number of clinically relevant Gram-negative pathogens, DsbA is the primary disulfide oxidoreductase responsible for catalyzing the formation of disulfide bonds in secreted and membrane-associated proteins. In this study, a putative B. pseudomallei dsbA gene was evaluated functionally and structurally and its contribution to infection assessed. Results: Biochemical studies confirmed the dsbA gene encodes a protein disulfide oxidoreductase. A dsbA deletion strain of B. pseudomallei was attenuated in both macrophages and a BALB/c mouse model of infection and displayed pleiotropic phenotypes that included defects in both secretion and motility. The 1.9 angstrom resolution crystal structure of BpsDsbA revealed differences from the classic member of this family Escherichia coli DsbA, in particular within the region surrounding the active site disulfide where EcDsbA engages with its partner protein E. coli DsbB, indicating that the interaction of BpsDsbA with its proposed partner BpsDsbB may be distinct from that of EcDsbA-EcDsbB. Innovation: This study has characterized BpsDsbA biochemically and structurally and determined that it is required for virulence of B. pseudomallei.Conclusion: These data establish a critical role for BpsDsbA in B. pseudomallei infection, which in combination with our structural characterization of BpsDsbA will facilitate the future development of rationally designed inhibitors against this drug-resistant organism

Crystal structure and site-directed mutagenesis of circular bacteriocin plantacyclin B21AG reveals cationic and aromatic residues important for antimicrobial activity

Plantacyclin B21AG is a circular bacteriocin produced by Lactiplantibacillus plantarum B21 which displays antimicrobial activity against various Gram-positive bacteria including foodborne pathogens, Listeria monocytogenes and Clostridium perfringens. It is a 58-amino acid cyclised antimicrobial peptide, with the N and C termini covalently linked together. The circular peptide backbone contributes to remarkable stability, conferring partial proteolytic resistance and structural integrity under a wide temperature and pH range. Here, we report the first crystal structure of a circular bacteriocin from a food grade Lactobacillus. The protein was crystallised using the hanging drop vapour diffusion method and the structure solved to a resolution of 1.8\ua0Å. Sequence alignment against 18 previously characterised circular bacteriocins revealed the presence of conserved charged and aromatic residues. Alanine substitution mutagenesis validated the importance of these residues. Minimum inhibitory concentration analysis of these Ala mutants showed that PheAla and TrpAla mutants displayed a 48- and 32-fold reduction in activity, compared to wild type.\ua0The LysAla mutant displayed the weakest activity, with a 128-fold reduction. These experiments demonstrate the relative importance of aromatic and cationic residues for the antimicrobial activity of plantacyclin B21AG and by extension, other circular bacteriocins sharing these evolutionarily conserved residues

Compound 10 demonstrated weak inhibitory activity.

<p><b>A.</b> Differential scanning fluorimetry profile with increasing concentrations of compound <b>10</b>. Similar to all other compounds tested, there was no significant shift in the unfolding temperature of EcDsbA up to 2 mM of compound <b>10</b>. <b>B.</b> ITC profile of EcDsbA titration by compound <b>10</b>, which shows no detectable binding under the conditions used (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133805#sec002" target="_blank">methods</a> for details). A similar outcome was found for the other 9 compounds. <b>C.</b> Compound <b>10</b> was the only one of the ten tested peptidomimetics that exhibited detectable activity in the DsbA assay, inducing a reduction in DsbA folding activity. <b>D.</b> Plotting the log of the peptidomimetic concentration against the rate of fluorescence increase measured in the enzyme assay allowed fitting of a sigmoidal curve and an estimated IC₅₀ value of ~1 mM for compound <b>10</b>. The positive control with no compound is shown as a white circle.</p

Comparison of the docked designed peptidomimetic with the EcDsbA-EcDsbB and PmDsbA-PWATCDS crystal structures.

<p>Calculated electrostatic surfaces of the enzymes are shown, with acidic regions in red, basic regions in blue and non-polar (hydrophobic) regions in white. Electrostatics cut-offs used are +/- 7.5 keV. <b>A.</b> Detail of the EcDsbA complex with EcDsbB from the crystal structure (PDB code 2ZUP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133805#pone.0133805.ref037" target="_blank">37</a>]) centred on the ⁹⁷YPSPFATCDFMVR¹⁰⁹ sequence of EcDsbB (in light blue) showing Phe101 (F101) binding in the EcDsbA hydrophobic groove (circled). <b>B.</b> Detail of the PmDsbAC30S:PWATCDS crystal structure (PDB code 4OD7) with PWATCDS in magenta. Residue Trp2 (W2) of the peptide binds in the PmDsbA hydrophobic groove (circled). <b>C.</b> Virtual screening identified compound <b>1</b> as a potential hit. Three optimal conformations of <b>1</b> are shown (in differing shades of green), in their predicted binding mode to the PmDsbAC30S hydrophobic groove. Potential hydrogen bonds between the morpholine moiety and DsbA Pro150, His32 and Asn162 are shown as yellow dashed lines.</p

Synthetic route for the tripeptide peptidomimetics.

<p>Synthetic route for the tripeptide peptidomimetics.</p

Surface properties of CtDsbA.

<p>Surface representation for CtDsbA of the catalytic (left) and non-catalytic (right) faces. The active site residues Cys-Ser-Ala-Cys are colored yellow and the nucleophilic cysteine sulfur highlighted in orange. Pockets formed on the posterior face of the protein between H1 and H3 (pocket 1) and the N-terminal unstructured region and H6 (pocket 2) are labeled. B. Electrostatic surface representation of CtDsbA. Views are oriented as above. Electrostatic surface potential is contoured between -5 (red) and +5 (blue) kT/e. The nucleophilic cysteine is annotated with an S.</p

Sequence alignment of DsbAs with two disulfide bonds.

<p>Sequence alignment of structurally characterised DsbAs with two disulfide bonds. Sequences were aligned using Clustal Omega and visualized using ESPript 3.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168485#pone.0168485.ref053" target="_blank">53</a>]. Secondary structural elements are shown for the structure of CtDsbA. Disulfide bonds are indicated with black connecting lines and labeled S-S. The single unpaired cysteine C70 is highlighted in yellow.</p

Chemical structures of the 10 peptidomimetic compounds synthesized and tested in this work.

<p>Compound <b>1</b> is the hit from the virtual screening from which derivatives <b>2</b>–<b>10</b> were designed.</p