Theoretical model of tandem repeat heparin-binding domain.

<p>The thrombospondin-related anonymous protein (TRAP) of the malaria parasite from PDB (code 1LSL) was used as a template for modeling the backbone of theoretical models. <b>(A)</b> The 3-D structure of TRAP itself is a modified triple β sheet, with 3 tryptophans (yellow) on one strand buried internal to the triple sheet. The guanidinium groups from arginines (white) of the neighboring strand intercalate the tryptophans to form stacked pi clouds. The tryptophan strand is a distorted β sheet with the sequence WxxWxxW, while the intercalation chain is a β sheet with the sequence QxRxRx. <b>(B)</b> In the 3-D model presented for the BAD-1 tandem repeat, the intercalation between tryptophans (yellow) and basic residues (white) takes place “proximal” to the disulfide bond. Histidine and lysine residues replace the arginines present in the TRAP structure (nitrogens, blue). Theoretical structures were constructed and energy minimized in Sybyl. <b>(C)</b> Depiction in 2-dimensions of possible configurations of WxxWxxW tryptophans and BxBxB basic residues as they might intercalate to form a heparin-binding cleft in proximity to bonded cysteine residues (horizontal blue line). In the “proximal” (left) and “distal” (center) models, these interactions would likely stabilize a repeating, anti-parallel, β-sheet secondary structure. In the third “hairpin” model (right), tandem repeats would run in long, anti-parallel stretches to form extended hairpin structures.</p

NMR determination of the structure of the tandem repeats.

<p><b>(A)</b>¹H-¹⁵N HSQC spectrum of TR4. Peaks arising from backbone amides of the representative repeat are labeled, while peaks from side chain N-H groups are indicated with smaller labels. Note that the four tryptophan side chains yield four distinctive peaks indicating distinct chemical environments. <b>(B)</b>¹H-¹⁵N HSQC spectrum of full length BAD-1 protein. <b>(C)</b>¹H-¹⁵N HSQC spectra of TR4 and BAD-1 overlaid to show that the chemical shifts for the amino acids of the TR4 repeats are similar to those of the full length BAD-1 repeats. <b>(D)</b> Structure of one tandem repeat loop and its tryptophan residues. We determined the structure of one repeat, focusing upon the loop created by the disulfide bond between the two universally conserved cysteines. The repeat forms two tightly folded turns followed by a short α-helix. All but one of the tryptophan residues are buried in the center of the tandem repeat fold. <b>(E)</b> Structure of one tandem repeat loop and its acidic residues. Negatively charged residues are uniformly oriented on the external surface of the loop. <b>(F)</b> Overlapping image of the top 20 structural predictions by CYANA. Variability is seen primarily in externalized side-chains (depicted using thin green lines). <b>(G)</b> Theoretical structure of the BAD-1 molecule. Model is based on energy minimization of the hinge regions and the fact that extensive steric hindrance between the tandem-repeat loops further limits flexibility. In this model, tandem repeats lie along a helical twist, with roughly 3.2 repeats describing one full turn. Individual tandem repeat structures are sequentially colored: pink, yellow, green, blue (and repeated).</p

Role of BAD-1 tandem repeats in virulence of <i>B. dermatitidis in vivo</i>.

<p>Mice received various strains of engineered <i>B. dermatitidis</i> yeasts (10⁴) intra-nasally in 25-µl PBS. Strains included wild-type (ATCC 26199), isogenic BAD-1 knockout (#55), strain #55 transformed to re-express BAD-1 (BAD-1-6H) and truncated forms of BAD-1 lacking 20 repeats (Trepeat20) or the C-terminus (ΔC-term). Mice infected with yeast expressing Trepeat20 showed significantly increased survival compared to controls. Mice receiving recombinant strains expressing the full complement of tandem repeats showed no significant alteration in survival compared to mice receiving wild-type 26199 yeast. All mice receiving strain #55 survived until the experiment was terminated five months post-infection.</p

Affinity of BAD-1 for heparin measured by SPR.

<p><b>(A)</b> BAD-1 binding to immobilized heparin monitored by surface plasmon resonance detection (SPR) using a Biorad Proteon XPR36. BAD-1 at the indicated concentrations was injected onto Biorad NLC neutravidin surface with biotinylated heparin immobilized to levels of 5 (circles) and 30 (squares) RUs. For clarity, only every 15th data point is shown. The solid lines are fits to the Langmuir binding model, on and off rates were fit to each sensogram but maximal response was fit to a single value for each immobilization level. The affinity was calculated from the rate constants to be 33±14 nM. <b>(B)</b> Heparin inhibition of BAD-1 binding to biotinylated heparin immobilized on a neutravidin surface. 0.375 µM BAD-1 with and without the addition of 3.75 µM heparin was injected onto the surface with heparin immobilized to levels of 30 (low), 59 (intermediate), and 96 (high) RU. Binding assays were performed using a buffer at physiological ionic strength (100 mM NaCl), similar to that in alveolar mucous (see Methods) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003464#ppat.1003464-Joris1" target="_blank">[16]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003464#ppat.1003464-Kozlova1" target="_blank">[17]</a>.</p

Influence of competitors on BAD-1 binding to heparin.

<p><b>(A)</b> Effect of a WxxW motif heparin-binding peptide on binding of BAD-1 (eFluor605) to immobilized heparin. “None” denotes BAD-1 binding to heparin agarose with no competitor. The WxxW peptide, or a mutant control peptide, was incubated with heparin resin at 1 mg/ml before addition of fluorescent BAD-1. Binding was quantitated by fluorescence units detected in a Filtermax F5 plate reader. Results are the mean ± SEM two experiments. <b>(B)</b> Effect of TR4 reduction on binding to heparin. Samples were incubated with resin directly or first boiled for 3 min in buffer alone or buffer with 5 mM DTT. TR4 has four copies of the BAD-1 tandem repeat. ΔCterm has all 41 repeats, but no C-terminal EGF-like domain. Binding was quantified by A280 measurement. Reduced TR4 bound significantly better than untreated TR4 or TR4 boiled without DTT (<b>*</b>, p<0.05). Results are the mean ± SEM of two experiments. <b>(C)</b> Effect of reduced TR4 on binding of BAD-1 (eFluor605) to immobilized heparin. BAD-1 binding to heparin agarose was quantified with or without pretreatment of resin with reduced TR4 as in panel C. BAD-1 binding was quantified by fluorescence units as above. Mannan resin is a background control. Heparin resin pre-treated with reduced TR4 at 0.2 and 0.1 mg/ml bound BAD-1 significantly better than untreated heparin resin (*, p<0.05). Soluble heparin significantly blocked binding of BAD-1 to both of these pre-treated resins (**, p<0.05).</p

Primary structure of BAD-1 tandem repeats.

<p><b>(A)</b> BAD-1 includes 31 highly conserved tandem repeats and 10 degenerate repeats, which make up 90% of the mature protein excepting the C-terminal EGF-like domain (103 amino acids) and 14 amino acids at the N-terminus. Universally conserved amino acids are highlighted. <b>(B)</b> Consensus of both universally conserved and highly conserved amino acids. <b>(C)</b> Sequence of the recombinant TR4 protein containing 4 identical repeats. Residues at each position represent the residues most commonly found in the corresponding positions of the native repeats.</p

BAD-1 binding of heparin.

<p><b>(A)</b> Percentage of 1×10⁶<i>B. dermatitidis</i> yeast that bound to wells coated with increasing concentrations of Matrigel. Control contained no Matrigel. <b>(B)</b> Inhibition of yeast binding to Matrigel by different dilutions of anti-BAD-1 antiserum. <b>(C)</b> BAD-1 binding of heparin is saturable. 100 µl of 0.1 mg/ml BAD-1 was applied to various bed volumes of heparin-agarose resin (400–1500 ng heparin/µl resin) in the presence of 50 mM, 100 mM, and 150 mM NaCl. Unbound BAD-1 was quantified by A280. <b>(D)</b> Binding of BAD-1 to other resins. BAD-1 (eFluor605) pulled down with heparin agarose resin produced a robust fluorescent signal (rightmost column-positive binding control). Binding of BAD-1 (eFluor605) to uncoated agarose resin and resins coated with BSA, hemoglobin or mannan was measured for comparison. Fluorescent BAD-1 bound better to heparin agarose than each control (*, p<0.05), and binding to control resins was insignificant (p>0.05). Inset: Fluorescent BAD-1 binding to resin surfaces analyzed by fluorescence microscopy. <b>(E)</b> Inhibition of BAD-1 binding to heparin resin by soluble heparin. Fluorescent BAD-1 was pre-incubated with increasing concentrations of soluble heparin before exposure to heparin-agarose resin. <b>(F)</b> Inhibition of BAD-1 binding to heparin agarose by alternate GAGs. 0.1 mg/ml fluorescent BAD-1 was pre-incubated with heparin, dermatan sulfate, chondroitin sulfate A, or hyaluronan for 20 min, followed by incubation with heparin-agarose for 30 min. Inhibition by heparin is significant vs. controls and other GAGs. *, p<0.05. Chondroitin sulfate A and hyaluronan are not significantly different from each other or controls. Dermatan sulfate inhibits BAD-1 binding only at 1 mg/ml, but not at lower concentrations. Results are the mean ± SEM of two to five experiments/panel.</p

Suppression of T cell activation mediated by BAD-1 binding of CD47.

<p><b>(A)</b> BAD-1 inhibition of CD69 expression is dependent on CD47. Jurkat T cells were activated by anti-CD3 antibody alone (5 µg/ml) or in the presence of BAD-1 or TSP1 (10 µg/ml) for 2 hours <i>in vitro</i>. RNA was isolated and relative CD69 mRNA expression determined by real-time PCR. <b>(B)</b> JinB8 T cells lacking CD47 were activated by anti-CD3 antibody alone or in the presence of BAD-1 or TSP1 for 2 hours <i>in vitro</i>. RNA was isolated and relative CD69 mRNA expression determined as above. <b>(C)</b> Un-transfected JinB8 cells and cells transfected with CD47 and CD47-S64A were activated by anti-CD3 antibody alone or in the presence of BAD-1 or TSP-1 for 2 hours <i>in</i> vitro. RNA was isolated and relative CD69 mRNA expression determined as above. Values are percent activation relative to stimulated cells ± SD of 4 experiments for data in panels A–C; *, p<0.05. <b>(D)</b> CD4⁺ T cells from 1807 TCR Tg mice were exposed to increasing amounts of BAD-1 for 90 minutes, washed and added to co-culture of BAD-1 null <i>B. dermatitidis</i> yeast and DC for 96 hours. After incubation, the T cells were analyzed by flow cytometry for expression of CD69. <b>(E)</b> BAD-1 (50 µg/ml) was incubated with 1807 cells at 37°C for 90 min and analyzed by FACS with anti-BAD-1 mAb conjugated to FITC. In top panel, the numbers are the mean fluorescence intensity of mAb binding to control-treated (grey) vs. BAD-1-treated (black) cells. After incubating 1807 Tg cells as above, the T cells were added to wells containing DC and <i>Blastomyces</i> cell wall/membrane antigen (10 µg/ml). After 24 hours the cells were stained for the activation markers shown and analyzed by FACS. <b>(F)</b> Supernates were collected from co-cultures in panel D and assayed by ELISA for IL-17A and IFN-γ. *, p<0.05. vs. control. Results are representative of 2–4 independent experiments for panels D and E.</p