Substrate affinity.

<p><b>A.</b> NADH binding to 50 µM apoWrbA determined by UV spectroscopy. Difference absorbance at 265 nm (see text) is plotted <i>vs.</i> [NADH]. The solid line is intended only to guide the eye and does not represent a fit to the data. <b>B.</b> NAD binding to 200 µM apoWrbA detected by ³¹P NMR. Spectra at 100, 200, 500, 1000 and 2000 µM NAD from bottom to top, respectively, are overlaid. The bracket with four arrows indicates the doublet pair characteristic of free NAD.</p

Substrate binding sites. A. NADH.

<p>View of the active site with NADH bound in the optimized position found by docking as described in the text. Green, molecular surface of holoWrbA calculated from the 2.05 Å crystal structure (PDB ID 3B6J) after removal of the FMN cofactor. Oxidized FMN is depicted as a skeletal model in atomic colors with cyan carbon, and docked NADH with white carbons for differentiation from FMN. Dashed lines represent the indicated distances in Å between nicotinamide C4 and each indicated electron acceptor site of FMN. <b>B. Mutual exclusivity of NADH and BQ.</b> Viewpoint of the binding cavity as in panel A but slightly zoomed out to better depict the steric environment of the full pocket. Translucent white indicates the molecular surface of NADH in the position identified by docking as in panel A; red indicates the molecular surface of BQ calculated from the 1.99 Å crystal structure of the BQ/WrbA complex (PDB ID 3B6K). The part of each substrate that is occluded by the other is represented by the overlap between the red and translucent white surfaces.</p

Product Inhibition^a.

a<p>Kinetic data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043902#pone.0043902.s003" target="_blank">Figure S3</a>.</p

Sedimentation velocity.

<p>Each panel shows the sedimentation velocity profile using the whole boundary g(s*) approach of Stafford <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043902#pone.0043902-Stafford1" target="_blank">[28]</a> for apoWrbA (black), WrbA+50 µM FMN (red), and WrbA+50 µM FMN+0.5 mM NAD (blue). A, 3 µM total protein (monomer) at 5°C; B, 3 µM total protein (monomer) at 20°C; C, 20 µM total protein (monomer) at 5°C; D, 20 µM total protein (monomer) at 20°C.</p

Steady-state kinetics.

<p>Initial velocity (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043902#s4" target="_blank">Methods</a>) is plotted <i>vs.</i> substrate concentration. <b>A.</b> NADH at constant [BQ] = 50 µM. <b>B.</b> BQ at constant [NADH] = 50 µM. <b>C.</b> DCPIP at constant [NADH] = 50 µM. Each plot depicts three temperature treatments of WrbA prior to assay (see text): squares, 5°C; triangles, 23°C; circles, 5°C after 23°C. Solid lines are intended only to guide the eye and do not represent fits to the data.</p

Ping-pong kinetics.

<p>Initial velocities were determined at 23°C to limit the reaction to a single kinetic phase as much as possible. <b>A.</b> Titration of NADH at [BQ] = 10 µM (open squares), 20 µM (triangles), 50 µM (circles), 100 µM (filled squares). <b>B.</b> Titration of BQ at [NADH] = 10 µM (open squares), 20 µM (triangles), 50 µM(circles), 100 µM(filled squares). Solid lines represent non-linear least-squares best fit of the Michaelis-Menten equation to the data points. The values of apparent Km and apparent Vmax returned from the fit are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043902#pone-0043902-t001" target="_blank">Table 1</a>.</p

Kinetic constants of WrbA^a.

a<p>Kinetic data are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043902#pone-0043902-g003" target="_blank">Figure 3</a>.</p>b<p>Errors are one standard deviation derived from triplicate measurements and propagated to the ratio Vmax/Km.</p

Effect of salt.

<p>Initial velocities at 5°C are plotted <i>vs.</i> substrate concentration. <b>A.</b> Titration of BQ at [NADH] = 100 µM with no salt (circles), 0.25 M NaCl (squares), and 0.5 M NaCl (triangles). <b>B.</b> Titration of DCPIP at [NADH] = 100 µM; symbols as in panel A. Solid lines are intended only to guide the eye and do not represent fits to the data.</p

Additional file 1 of Bispecific T cell-engager targeting oncofetal chondroitin sulfate induces complete tumor regression and protective immune memory in mice

Additional file 1: Sup. Fig. 1. (A) ELISA showing binding of V-aCD3Mu (Coupled)(Kd = 38.8, Bmax = 3.31), rVAR2 (Kd and Bmax not determined), and V-aCD3Mu (Fused)(Kd = 14.2, Bmax = 3.36) to CSPG on a decorin backbone. Data is representative of a minimum of two separate experiments. (B) Solid 4T1 tumors 50-100 mm3 in size were treated with either PBS (n=5), V-aCD3Mu (Coupled) + CpG (n=8), or V-aCD3Mu (Fused) + CpG (n=8) on day 10, 12, 14, and 17 after tumor injection. Numbers in parentheses indicate the number of animals with complete tumor regression out of all mice in the group. Sup. Fig. 2. (A) Gating strategy on splenocytes and PBMCs in flow cytometry used to determine binding of rVAR2, aCD3Mu, V-aCD3Mu, aCD3Hu, and anti-V5 antibodies to T cells and non-T cell splenocytes/PBMCs. The gating is single cells lymphocytes live cells CD4+ and/or CD8+ cells as T cells and CD4-CD8- cells as non-T cells. The geometric MFI of the anti-penta-HIS antibodies conjugated to Alexa Flour 488 was then used to evaluate the binding of the HIS-tagged proteins. (B) Binding of aCD3Mu (Kd = 4.96, Bmax = 1.05), rVAR2 (Kd = NR, Bmax = 0.46), and V-aCD3Mu (Kd = 1.24, Bmax = 3.38) to murine recombinant CD3 in ELISA with aCD4Mu as a negative control (left). Means and standard deviations are shown. Right pane shows CSA inhibition of binding at 120 nM (right). Each dot represents one data point. Sup. Fig. 3. Cytokines measured from 4T1 and splenocyte co-culture supernatants using ELISA. Mouse splenocytes were incubated with 4T1 cancer cells together with 200 nM of the indicated protein. Sup. Fig. 4. (A) Survival curves for mice with indicated tumors treated as described in Fig. 4. The cut-off for all Kaplan-Meier plots is a tumor volume of $$\ge$$ ≥ 400 mm3. Mice were censored if they had to be excluded from the study prematurely due to reasons other than tumor size. Log-rank test was used for statistical analysis. *p < 0.05. (B) Bioluminescence in vivo imaging of C57BL/6 mice following orthotopic implantation of 5x104 Luciferase+ primary pancreatic cancer cells (CHX2000) derived from KPC mice (LSL-KrasG12D/+; p53f/f; Pdx1-Cre). Sup. Fig. 5. (A-C) Survival curves for mice treated as described in Fig. 5. The cut-off for all Kaplan-Meier plots is a tumor volume of $$\ge$$ ≥ 400 mm3. Mice were censored if they had to be excluded from the study prematurely due to reasons other than tumor size. Log-rank test was used for statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Sup. Fig. 6. (A) Treatment schedule until day 14 when spleens and tumors were harvested for flow cytometry and the subsequent gating strategy on splenocytes to evaluate different cell types in C-D. (B) Percentage of live cells relative to the PBS group in the spleen. Both CD8+ and CD4+ T cells that are CD69+, CD44hi, CD8+CD25+, or Tregs are shown. (C) UMAPs of splenocytes from all four treatment groups with clustering performed in ClusterExplorer. Cell types in clusters are explained below. Statistics were performed using one-way ANOVA with Dunnett’s post hoc test for comparison of all treatment groups to the PBS group. P values are indicated if significant or important for reading the figure. (D) Correlations between the tumor size and �8+CD69+ (p=0.67) and �4+CD69+ (p=0.14) of all live single cells in the tumor evaluated by simple linear regression. Sup. Fig. 7. Binding of mouse antibodies to 4T1 cells and B16-F10 cells in flow cytometry. Serum from C57BL/6 mice treated as described in materials and methods was diluted as illustrated on the figure and incubated with 200.000 4T1 or B16-F10 cells. Soluble CSA was added if indicated for 1 hour before detection with an anti-mouse IgG antibody conjugated to FITC

Quality of the cryoEM density.

(a) ID1 loop: N505 –K522; DBL2: L905 –N908, K914 –W917, Y958 –A963; DBL4: E1614 –R1617, K1872 –E1875 (b) Heavy chain CDR loops, CDR–H1: D47 –G52; CDR–H2: I70 –K77; CDR–H3: R117 –N131. (c) Light chain CDR loops, CDR–L1: E46 –N50; CDR–L2: I67 –A70; CDR–L3: Q108 –A116. The volume surface is zoned at 2.5Å in UCSF ChimeraX for all the regions. (PDF)</p