The Primed Ebolavirus Glycoprotein (19-Kilodalton GP1,2): Sequence and Residues Critical for Host Cell Binding▿ †

Entry of ebolavirus (EBOV) into cells is mediated by its glycoprotein (GP1,2), a class I fusion protein whose structure was recently determined (J. E. Lee et al., Nature 454:177-182, 2008). Here we confirmed two major predictions of the structural analysis, namely, the residues in GP1 and GP2 that remain after GP1,2 is proteolytically primed by endosomal cathepsins for fusion and residues in GP1 that are critical for binding to host cells. Mass spectroscopic analysis indicated that primed GP1,2 contains residues 33 to 190 of GP1 and all residues of GP2. The location of the receptor binding site was determined by a two-pronged approach. We identified a small receptor binding region (RBR), residues 90 to 149 of GP1, by comparing the cell binding abilities of four RBR proteins produced in high yield. We characterized the binding properties of the optimal RBR (containing GP1 residues 57 to 149) and then conducted a mutational analysis to identify critical binding residues. Substitutions at four lysines (K95, K114, K115, and K140) decreased binding and the ability of RBR proteins to inhibit GP1,2-mediated infection. K114, K115, and K140 lie in a small region modeled to be located on the top surface of the chalice following proteolytic priming; K95 lies deeper in the chalice bowl. Combined with those of Lee et al., our findings provide structural insight into how GP1,2 is primed for fusion and define the core of the EBOV RBR (residues 90 to 149 of GP1) as a highly conserved region containing a two-stranded β-sheet, the two intra-GP1 disulfide bonds, and four critical Lys residues

<i>Pfi</i>t Is a Structurally Novel Crohn's Disease-Associated Superantigen

<div><p>T cell responses to enteric bacteria are important in inflammatory bowel disease. I2, encoded by the <i>pfi</i>T gene of <i>Pseudomonas fluorescens</i>, is a T-cell superantigen associated with human Crohn's disease. Here we report the crystal structure of <i>pfi</i>T at 1.7Å resolution and provide a functional analysis of the interaction of <i>pfi</i>T and its homolog, PA2885, with human class II MHC. Both <i>pfi</i>T and PA2885 bound to mammalian cells and stimulated the proliferation of human lymphocytes. This binding was greatly inhibited by anti-class II MHC HLA-DR antibodies, and to a lesser extent, by anti HLA-DQ and DP antibodies, indicating that the binding was class II MHC-specific. GST-<i>pfi</i>T efficiently precipitated both endogenous and <i>in vitro</i> purified recombinant HLA-DR1 molecules, indicating that <i>pfi</i>T directly interacted with HLA-DR1. Competition studies revealed that <i>pfi</i>T and the superantigen <i>Mycoplasma arthritidis</i> mitogen (MAM) competed for binding to HLA-DR, indicating that their binding sites overlap. Structural analyses established that <i>pfi</i>T belongs to the TetR-family of DNA-binding transcription regulators. The distinct structure of <i>pfi</i>T indicates that it represents a new family of T cell superantigens.</p></div

Binding of <i>pfi</i>T and PA2885 to human PBMC is dependent on the class II MHC HLA-DR.

<p>(<b>A</b>) Analysis of binding of FITC-labeled control SAg MAM to CD19⁺ human lymphocytes, in the presence of various anti-class II MHC and control antibodies. (<b>B</b>) Binding profile of FITC-labeled <i>pfi</i>T to CD19⁺ human lymphocytes. (<b>C</b>) Binding profile of FITC-labeled PA2885 to CD19⁺ human lymphocytes.</p

Crystal structure of <i>pfi</i>T.

<p>(<b>A</b>) Cartoon representation of crystal structure of native <i>pfi</i>T monomer. Secondary structural elements were labeled. (<b>B</b>) Cartoon representation of crystal structure of the <i>pfi</i>T dimer of the Se-Met <i>pfi</i>T crystal. The N-terminal α1 helix that is missing in the native <i>pfi</i>T structure and dimer interface helices were labeled. (<b>C</b>) Comparison of the dimer of Se-Met <i>pfi</i>T (green and red) with that of native <i>pfi</i>T (blue) reconstituted through crystallographic symmetry in the C2 crystal form. (<b>D</b>) Superposition of <i>pfi</i>T (red) to a putative TetR repressor (blue) from <i>Vibrio parahaemolyticus</i> (PDB code: 3HE0). (<b>E</b>) Superposition of <i>pfi</i>T dimer to that of the QacR-DNA complex (PDB: 1JT0) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003837#ppat.1003837-Schumacher2" target="_blank">[37]</a>. <i>pfi</i>T was colored as red and green for the two monomers of the dimer; the QacR dimer was colored yellow. (<b>F</b>) Structure comparison of <i>pfi</i>T with QacR at the QacR DNA-binding site. Residues that are important for DNA-binding were labeled in black (QacR) and green (<i>pfi</i>T), and presented in stick representation, with oxygen in red, nitrogen in blue, and carbon either in green (<i>pfi</i>T) or in yellow (QacR). (<b>G</b>) The electrostatic surface potential of <i>pfi</i>T at the putative DNA-binding site, with blue and red regions indicating positive and negative electrostatic regions, respectively. This figure was made with GRASP <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003837#ppat.1003837-Nicholls1" target="_blank">[57]</a>. (<b>H</b>) Structure-based alignment of sequences of <i>pfi</i>T and TetR members (TetR <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003837#ppat.1003837-Orth1" target="_blank">[32]</a>, QacR <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003837#ppat.1003837-Schumacher2" target="_blank">[37]</a>, DesT <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003837#ppat.1003837-Miller1" target="_blank">[33]</a>) with known three dimensional structures of protein-DNA complexes. Residues that make direct interactions with DNA elements were shaded. Residues were colored according to the extent of their sequence conservation: strictly conserved (red); >50% conservation (green); and not conserved (<50%) (black).</p

Analytical ultracentrifugation analysis of <i>pfi</i>T binding to recombinant HLA-DR1/HA complex.

<p>(<b>A</b>) Sedimentation coefficient distributions of <i>pfi</i>T alone at 3.6 µM concentration. (<b>B</b>) Sedimentation equilibrium analysis of <i>pfi</i>T. Representative absorbance distributions were shown for sedimentation equilibrium of <i>pfi</i>T at 18 µM (left panel) and 9 µM (right panel) at 20°C at rotor speeds of 20,000 rpm (black solid square), 25,000 rpm (red solid circle), and 30,000 rpm (green solid triangle). Distributions were analyzed as part of a global fitting to the absorbance data at multiple loading concentrations and multiple speeds. <i>Solid lines</i> are the global best-fit distributions using a reversible monomer and dimer model with SEDPHAT <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003837#ppat.1003837-Vistica1" target="_blank">[27]</a>. (<b>C</b>) Sedimentation equilibrium analysis of <i>pfi</i>T binding to the HLA-DR1/HA complex. Absorbance distributions were shown for sedimentation equilibrium of the <i>pfi</i>T-DR1 complex at different molar ratios at 20°C at rotor speeds of 20,000 rpm (black solid square), 25,000 rpm (red solid circle), and 30,000 rpm (green solid triangle). Data were analyzed as in (<b>B</b>).</p

Soluble recombinant <i>pfi</i>T and PA2885 stimulate the activation of lymphocytes.

<p>(<b>A</b>) SDS-PAGE analysis of purified recombinant <i>pfi</i>T and PA2885 proteins. Lane 1, purified <i>pfi</i>T-GST fusion protein; Lanes 2 and 3, molecular weight standard; Lane 4, purified <i>pfi</i>T; Lane 5, purified PA2885. (<b>B</b>) Stimulation of murine splenocytes by <i>pfi</i>T and control SAg SEB. (<b>C</b>) Stimulation of human PBMC by <i>pfi</i>T, <i>pfi</i>T-GST fusion protein, and control SAg SEB. (<b>D</b>) Proliferation profiles of human T cells labeled with CFSE, stimulated by <i>pfi</i>T, PA2885, and control SAgs.</p

<i>Pfi</i>T directly interacts with the class II MHC HLA-DR.

<p>(<b>A</b>) Western blot analysis of interactions between <i>pfi</i>T and HLA-DR. <i>pfi</i>T-GST, control SAg GST-MAM, and negative control GST were used in GST pull down assay of the cell lysates of class II MHC HLA-DR+ Raji cells and purified recombinant HLA-DR expressed in <i>E. coli</i>. (<b>B</b>) <i>pfi</i>T binding to HLA-DR is dose dependent. Purified recombinant HLA-DR/HA complex was used. (<b>C</b>) Curve fitting to determine the affinity of <i>pfi</i>T binding to recombinant HLA-DR/HA complex.</p

The binding site on HLA-DR for <i>pfi</i>T overlaps with that for MAM.

<p>(<b>A</b>) <i>pfi</i>T competes GST-MAM to bind HLA-DR in a dose-dependent manner. (<b>B</b>) MAM competes GST-<i>pfi</i>T to bind HLA-DR in dose-dependent manner. (<b>C</b>) Curve fitting to determine the IC₅₀ (concentration required for competitors to inhibit 50% binding) for <i>pfi</i>T inhibition of MAM binding to HLA-DR or versus visa. Experiment was performed in duplicate.</p