A Model Study for Constructing the DEF-Benzoxocin Ring System of Menogaril and Nogalamycin via a Reductive Heck Cyclization

A novel reductive Heck cyclization approach was developed in order to construct a model DEF-benzoxocin ring system that is present in nogalamycin, menogaril, and related anthracycline antitumor antibiotics

Solid-Phase Synthesis of Lysobactin (Katanosin B): Insights into Structure and Function

The solid phase synthesis of the cyclic depsipeptide antibiotic lysobactin is described. The natural product was synthesized via a linear approach using mostly an Fmoc-strategy solid phase peptide synthesis (SPPS) with a single purification. A lysobactin analog has also been synthesized displaying nanomolar membrane disruption activity not seen with the natural product

An Assembly-Activating Site in the Hepatitis B Virus Capsid Protein Can Also Trigger Disassembly

The Hepatitis B Virus (HBV) core protein homodimers self-assemble to form an icosahedral capsid that packages the viral genome. Disassembly occurs in the nuclear basket to release the mature genome to the nucleus. Small molecules have been developed that bind to a pocket at the interdimer interface to accelerate assembly and strengthen interactions between subunits; these are under development as antiviral agents. Here, we explore the role of the dimer–dimer interface by mutating sites in the drug-binding pocket to cysteine and examining the effect of covalently linking small molecules to them. We find that ligands bound to the pocket may trigger capsid disassembly in a dose-dependent manner. This result indicates that, at least transiently, the pocket adopts a destabilizing conformation. We speculate that this pocket also plays a role in virus disassembly and genome release by binding ligands that are incompatible with virus stability, “unwanted guests.” Investigating protein–protein interactions, especially large protein polymers, offers new and unique challenges. By using an engineered addressable thiol, we provide a means to examine the effects of modifying an interface without requiring drug-like properties for the ligand

Design and Synthesis of a Stable Oxidized Phospholipid Mimic with Specific Binding Recognition for Macrophage Scavenger Receptors

Macrophage scavenger receptors appear to play a major role in the clearance of oxidized phospholipid (OxPL) products. Discrete peptide–phospholipid conjugates with the phosphatidylcholine headgroup have been shown to exhibit binding affinity for these receptors. We report the preparation of a water-soluble, stable peptide–phospholipid conjugate (<b>9</b>) that possesses the necessary physical properties to enable more detailed study of the role(s) of OxPL in metabolic disease

Electroreductive Dimerization of Coumarin and Coumarin Analogues at Carbon Cathodes

Electrochemical reduction of coumarin (<b>1</b>), 6-methylcoumarin (<b>2</b>), 7-methylcoumarin (<b>3</b>), 7-methoxycoumarin (<b>4</b>), and 5,7-dimethoxycoumarin (<b>5</b>) at carbon cathodes in dimethylformamide containing 0.10 M tetra-<i>n</i>-butylammonium tetrafluoroborate has been investigated by means of cyclic voltammetry and controlled-potential (bulk) electrolysis. Cyclic voltammograms for reduction of <b>1</b>–<b>5</b> exhibit two irreversible cathodic peaks: (a) the first peak arises from one-electron reduction of the coumarin to form a radical–anion intermediate, which is protonated by the medium to give a neutral radical; (b) although most of this radical undergoes self-coupling to yield a hydrodimer, reduction of the remaining radical (ultimately to produce a dihydrocoumarin) causes the second cathodic peak. At a potential corresponding to the first voltammetric peak, bulk electrolysis of <b>1</b>–<b>5</b> affords the corresponding hydrodimer as a mixture of <i>meso</i> and <i>dl</i> diastereomers. Although the <i>meso</i> form dominates, the <i>dl</i>-to-<i>meso</i> ratio varies, due to steric effects arising from substituents on the aromatic ring. Electroreduction of an equimolar mixture of <b>1</b> and <b>4</b> gives, along with the anticipated symmetrical hydrodimers, an unsymmetrical product derived from the two coumarins. A mechanistic scheme involving both radical–anion and radical intermediates is proposed to account for the formation of the various products

Models for PG synthesis in <i>S. pneumoniae</i>.

<p>In this model, a large membrane PG assembly complex (Yin Yang circle) contains both the septal (red) and the peripheral (orange) PG assembly machineries. The two transpeptidases PBP2x and PBP2b (noted 2x and 2b) and the two lipid-flippases FtsW and RodA (noted W and A) are indicated in green and blue, respectively. Non-phosphorylated forms of DivIVA and other StkP substrates are required for cell elongation and thus peripheral PG synthesis. GpsB is not <i>per se</i> involved in the production of the cross-wall, but is required at the septum to localize StkP (light green oval), to allow the phosphorylation of StkP substrates including DivIVA and to favor production of septal PG by down-regulating peripheral PG synthesis. The paralogs GpsB (pink oval) and DivIVA (purple oval) constitute a molecular switch that connects, together with EzrA (green oval), the Z-ring with the PG assembly complex. StkP kinase activity, counterbalanced by the phosphatase PhpP (yellow oval) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004275#pgen.1004275-Beilharz1" target="_blank">[15]</a> and triggered by GpsB, modulates the function of a set of proteins (dashed ovals) including DivIVA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004275#pgen.1004275-Fleurie1" target="_blank">[14]</a>. The StkP/DivIVA/GpsB triad is thus proposed to orchestrate and to finely tune production of septal and peripheral peptidoglycan synthesis responsible for the ovoid-shape of pneumococcus.</p

Localization of DivIVA in WT and Δ<i>gpsB</i> cells.

<p>(A) DivIVA-GFP localization in WT and Δ<i>gpsB</i> cells. Phase contrast (left), GFP fluorescent signal (middle) and overlays (right) between phase contrast (red) and GFP (green) images are shown. (B) Co-localization of FtsZ-RFP (red) and DivIVA-GFP (green) in WT and Δ<i>gpsB</i> cells. Overlays between phase contrast (gray), GFP (green), and RFP (red) are shown. Cells were grown to exponential phase in THY medium at 37°C. Arrows show helical organization of DivIVA-GFP and FtsZ-RFP. Scale bar, 5 µm. DivIVA-GFP and FtsZ-GFP are the only source of FtsZ and DivIVA in cells. <i>ftsZ-gfp</i> and <i>divIVA-gfp</i> substitute the native <i>ftsZ</i> and <i>divIVA</i> genes at their chromosomal locus, respectively.</p

Alignment for GpsB and DivIVA proteins from several bacteria.

<p>(A) Multiple sequence alignments of GpsB and DivIVA sequences from streptococci and Gram-positive bacteria. Protein sequences similar to that of pneumococcus GpsB and DivIVA were identified by BLAST searches and aligned using CLUSTALW. Spn: <i>S. pneumoniae</i>; Sag: <i>S. agalactiae</i>, Bsu: <i>B. subtilis</i>; Sta: <i>S. aureus</i>, Mtb: <i>M. tuberculosis</i>; Sco: <i>S. coelicolor</i>. Yellow highlights the potential coiled-coil motifs retrieved from UniProtKB/Swiss-Prot:Q8CWP9 and UniProtKB/Swiss-Prot:C1CIN3 entry annotations for Spn-DivIVA (residues 34–135 and 199–236) and Spn-GpsB (36–63) respectively. The PF05103 PFAM DivIVA family signatures are mapped as green open boxes for DivIVA and GpsB. When identified, phosphorylation sites are red boxed. The <i>S. coelicolor</i> DivIVA phosphopeptide containing unidentified phosphorylation sites are highlighted in orange letters. Identical residues are in pink letters and positions showing conservation of similar residues are in blue. Dots indicate gaps introduced in sequences during alignment computation. The figure was rendered with the ESPript server <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004275#pgen.1004275-Gouet1" target="_blank">[54]</a>. (B) Multiple sequence alignments of GpsB sequences from streptococci. Protein sequences were aligned using CLUSTALW. Spy: <i>S. pyogenes</i>; Sag: <i>S. agalactiae</i>, Smu: <i>S. mutans</i>; Sth: <i>S. thermophylus</i>; Ssa: <i>S. salivarius</i>; Spn: <i>S. pneumoniae</i>; Smi: <i>S. mitis</i>, Sgo: <i>S. gordonii</i>. The PFAM PF05103 DivIVA family signatures are mapped as green boxes. Yellow highlights the potential coiled-coil motifs retrieved from UniProtKB/Swiss-Prot:C1CIN3 entry annotations for Spn-GpsB (36–63). The phosphothreonine identified for <i>S. agalactiae</i> GpsB is red boxed. Glutamic acids possibly mimicking threonine phosphorylation are black boxed with white letters. Identical residues are in pink letters and positions showing conservation of similar residues are in blue. Dots indicate gaps introduced in sequences during alignment computation. The figure was rendered with the ESPript server <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004275#pgen.1004275-Gouet1" target="_blank">[54]</a>.</p

Interplay of GpsB, DivIVA and StkP.

<p>(A) Western immunoblot of whole-cell lysates from the wild type (WT), Δ<i>stkP</i>, <i>gpsB::gfp-gpsB</i>, Δ<i>gpsB</i>, Δ<i>divIVA</i> and Δ<i>gpsB</i>Δ<i>divIVA</i> cells grown in THY at 37°C probed with anti-phosphothreonine antibodies. The same amounts (25 µg) of proteins were loaded in all gel lanes. Arrow indicates the signal observed around 15 kDa. The phosphorylation signal for DivIVA and StkP are indicated. (B) Western immunoblot of whole-cell lysates from wild type (WT) or <i>gpsB::gfp-gpsB</i> cells probed with anti-GFP antibodies. Purified GFP is used as control. Arrow indicates the signal observed for GFP-GpsB. (C) StkP localization using a GFP N-terminal fusion in WT, Δ<i>gpsB</i>, Δ<i>divIVA</i> and Δ<i>gpsB</i>Δ<i>divIVA</i> cells. GFP (green) and phase-contrast (grey) images were taken from a typical field of exponentially grown cells in THY at 37°C. Merged pictures (lower panels) show the overlay of StkP (green) and phase contrast images (red). Scale bar, 5 µm. (D) Cell morphology of <i>stkP-K42M</i> cells deficient for DivIVA or GpsB expression. Cells producing a kinase dead-form of StkP (<i>stkP-K42M</i>, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004275#pgen.1004275-Fleurie1" target="_blank">[14]</a>) were deleted either for <i>divIVA</i> or <i>gpsB</i> resulting thus in Δ<i>divIVA</i>-<i>stkP-K42M</i> and Δ<i>gpsB</i>-<i>stkP-K42M</i> strains, respectively. Phase contrast microscopy (upper row) and FM4–64 membrane staining (lower row) images of Δ<i>divIVA</i>-<i>stkP-K42M</i> (left panel) and Δ<i>gpsB</i>-stkP-K42M (right panel) exponentially growing cells at 37°C in THY medium. Scale bar, 5 µm.</p

Morphology of WT, Δ<i>divIVA</i>, Δ<i>gpsB</i> and Δ<i>divIVA</i>Δ<i>gpsB</i> cells.

<p>(A) Phase contrast microscopy (lower panel) and FM4–64 membrane staining (upper panel) images of WT, Δ<i>divIVA</i>, Δ<i>gpsB</i> and Δ<i>divIVA</i>Δ<i>gpsB</i> exponentially growing cells at 37°C in THY medium. Scale bar, 5 µm. (B) Scanning electron micrograph of WT, Δ<i>divIVA</i>, Δ<i>gpsB</i> and Δ<i>divIVA</i>Δ<i>gpsB</i> cells. Scale bar, 1 µm. (C) Transmission electron micrograph of WT, Δ<i>divIVA</i>, Δ<i>gpsB</i> and Δ<i>divIVA</i>Δ<i>gpsB</i> cells. Scale bar, 1 µm. Asterisks indicate defective septal initiations in staggered rows in the Δ<i>gpsB</i> cell.</p