Interaction of the ^FtDHPS module with Compound 1.

<p>(A) Schematic comparison between the scaffolds of Compound 1 and DHP-PP. Compound 1 comprises a pterin-like core and is missing half of the B-ring as highlighted in orange. (B) Stereo view of Compound 1 (orange) bound within the pterin pocket of the TIM-barrel. Residues that make van der Waals and hydrogen-bond contacts are labeled and shown as pink sticks. The <i>F</i>o-<i>F</i>c simulated-annealing omit electron density for Compound 1 is shown as a blue mesh contoured at 3.5 σ.</p

The overall structure of the HPPK-DHPS bifunctional enzyme from <i>Francisella tularensis</i>.

<p>(A) A stereo view of the overall fold and domain organization showing the secondary structure elements within each module. Each element is labeled with the prefixes ‘H’ and ‘D’ to reflect their locations in the HPPK (blue) and DHPS (purple) domains, respectively. The N- and C-termini and the linker region (green) are labeled. Note that helix Dα8 in the canonical DHPS TIM-barrel is missing. (B) A surface representation of the view shown in (A) that highlights the position of the domain linker and the cleft within the DHPS module corresponding to the missing Dα8 TIM-barrel α-helix.</p

The primary structure of the HPPK-DHPS bifunctional enzyme from <i>Francisella tularensis</i> and its homology to other HPPK and DHPS enzymes.

<p>The organisms shown are <i>Francisella tularensis</i> (Ft), <i>Saccharomyces cerevisiae</i> (Sc), <i>Yersinia pestis</i> (Yp), <i>Escherichia coli</i> (Ec) and <i>Bacillus anthracis</i> (Ba), and numbering is with respect to the Ft enzyme. Secondary structure elements and key structural regions are labeled according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014165#pone-0014165-g003" target="_blank">Fig. 3A</a>. Strictly conserved regions are blocked in red, and conserved regions are boxed. Important loop regions are highlighted and labeled according to their domain association. (A) Multiple sequence alignment of the HPPK module. Residues that contribute to substrate binding are shown as blue triangles. The conserved motif that binds Mg²⁺ is shown as gray circles within blue triangles. (B) Alignment of the DHPS module. The inter-domain linker regions of <i>F. tularensis</i> and <i>S. cerevisiae</i> are highlighted in green and the corresponding β-hairpin of monofunctional DHPS is highlighted in orange. Residues that interact with substrates are indicated as purple triangles. Residues known to contribute to sulfonamide drug resistance are indicated by red circles. The missing Dα8 helix at the C-terminus is highlighted in purple. Sequence alignments were performed using ClustalW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014165#pone.0014165-Thompson1" target="_blank">[39]</a> and analyzed using ESPript2.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014165#pone.0014165-Gouet1" target="_blank">[54]</a>.</p

Data Collection and Refinement Statistics.

<p>*Data were collected from a single crystal. Values in parentheses are for the highest-resolution shell.</p>a<p>R_free was calculated using 5% of the reflections.</p

Analytical ultracentrifugation of the HPPK-DHPS bifunctional enzyme from <i>Francisella tularensis</i>.

<p>(A) The sedimentation velocity profiles (fringe displacement) were fitted to a continuous sedimentation coefficient distribution model c(s). The experiment was conducted at a loading protein concentration of 0.69 mg/ml in at 20°C and at a rotor speed of 60,000 rpm. (B) Absorbance scans at 280 nm at equilibrium are plotted <i>versus</i> the distance from the axis of rotation. The protein was centrifuged at 4°C for at least 24 h at each rotor speed of 15 k (red), 22 k (blue) and 27 k (black) rpm. The <i>solid lines</i> represent the global nonlinear least squares best-fit of all the data sets to a monomer-dimer self-association model with a very weak K_D (2.7 mM). The loading protein concentration was 20 µM and the r.m.s. deviation for this fit was 0.0037 absorbance units.</p

Fab and FcR binding.

<p>(<b>A</b>) Linear cross-clade epitope mapping of 7B2 IgG1_AAA by peptide microarray. FcR binding (response units), on-rate (ka) and off rate (kd) by Surface Plasmon Resonance (SPR) of 7B2 IgG1_AAA. (<b>B</b>) Fine mapping of the 7B2 epitope within the gp41 immunodominant loop. Top Graph shows the binding response at saturation (~140 seconds after starting injection of 7B2 Fab) of each Ala-substituted gp41_596-606 peptide normalized to wild type and the middle graph shows the normalized off-rate of the same peptides. Data are representative of at least two measurements on adjacent spots in the same sensor chip. Residues that are part of the 7B2 epitope are colored in orange. The Lys601Ala mutant peptide is highlighted in green since it gave a higher binding response and a decreased off-rate. Bottom graph is an example of sensogram showing 7B2 Fab binding to WT and select Alanine mutant gp41_596-606 peptides that were used to generate the top and middle graphs. (<b>C</b>) Binding between 7B2 and gp41 peptides in standard and reducing conditions. (<b>D</b>) The structure of the 7B2 Fab-gp41 peptide complex shows detailed polar interactions. Hydrogen bonds between functional groups in the peptide and the heavy chain of the Fab are indicated. (<b>E</b>) Comparison of the gp41 ID loop from our structure (far left) against its structure obtained from NMR (middle left) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005042#ppat.1005042.ref038" target="_blank">38</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005042#ppat.1005042.ref039" target="_blank">39</a>] and its conformation as shown in the BG505.SOSIP.664 structure (middle right) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005042#ppat.1005042.ref015" target="_blank">15</a>] superimposed against the 7B2 paratope. A superposition of all three ID conformations (far right) highlights the conformational variability of this region.</p

Binding rate constants and affinities of rhesus FcγR3 binding to mAbs.

<p>Antibodies were measured for rhesus FcR binding by surface plasmon resonance (SPR). Mean and standard deviation from 3 independent assays are indicated where available.</p><p>Binding rate constants and affinities of rhesus FcγR3 binding to mAbs.</p

7B2 IgG1_AAA, A32 IgG1_AAA and CH22 IgG1_AAA mAb concentrations in (A) plasma and (B) rectal secretions.

<p>Concentrations of mAb were measured by a binding assay with the infused antibody as a control for calculating concentration equivalents of Ab binding to Env protein (μg/ml). Visible red blood cells in the rectal weck elutions were observed at time points post infusion for some animals.</p

Data collection and refinement statistics for the 7B2 Fab-gp41 _596–606 structure.

<p>^a The crystal had two Fab-peptide complexes in the asymmetric unit. The dataset came from a single crystal.</p><p>^b Values in parentheses are for the highest resolution shells.</p><p>Data collection and refinement statistics for the 7B2 Fab-gp41 _596–606 structure.</p

mAb concentrations at time of challenge.

<p>The average and range of antibody concentrations in plasma and rectal secretions from the PK study are indicated for the times that the subsequent infusion-challenge study was performed.</p><p>mAb concentrations at time of challenge.</p