Pfit is a structurally novel Crohn's disease-associated superantigen

Abstract

T cell responses to enteric bacteria are important in inflammatory bowel disease. I2, encoded by the pfiT gene of Pseudomonas fluorescens, is a T-cell superantigen associated with human Crohn's disease. Here we report the crystal structure of pfiT at 1.7Å resolution and provide a functional analysis of the interaction of pfiT and its homolog, PA2885, with human class II MHC. Both pfiT 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-pfiT efficiently precipitated both endogenous and in vitro purified recombinant HLA-DR1 molecules, indicating that pfiT directly interacted with HLA-DR1. Competition studies revealed that pfiT and the superantigen Mycoplasma arthritidis mitogen (MAM) competed for binding to HLA-DR, indicating that their binding sites overlap. Structural analyses established that pfiT belongs to the TetR-family of DNA-binding transcription regulators. The distinct structure of pfiT indicates that it represents a new family of T cell superantigens.

Figure 1. Soluble recombinant pfiT and PA2885 stimulate the activation of lymphocytes.

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

Figure 2. Analysis of binding of ¹²⁵I-lebdeled superantigens to murine splenocytes and human PBMC.

(A) MAM (control SAg); (B) pfiT; (C) PA2885. Experiments were performed in triplicate in the presence or absence of “cold” protein competitors.

Figure 3. Binding of pfiT and PA2885 to human PBMC is dependent on the class II MHC HLA-DR.

(A) 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) Binding profile of FITC-labeled pfiT to CD19⁺ human lymphocytes. (C) Binding profile of FITC-labeled PA2885 to CD19⁺ human lymphocytes.

Figure 4. PfiT directly interacts with the class II MHC HLA-DR.

(A) Western blot analysis of interactions between pfiT and HLA-DR. pfiT-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 E. coli. (B) pfiT binding to HLA-DR is dose dependent. Purified recombinant HLA-DR/HA complex was used. (C) Curve fitting to determine the affinity of pfiT binding to recombinant HLA-DR/HA complex.

Figure 5. Analytical ultracentrifugation analysis of pfiT binding to recombinant HLA-DR1/HA complex.

(A) Sedimentation coefficient distributions of pfiT alone at 3.6 µM concentration. (B) Sedimentation equilibrium analysis of pfiT. Representative absorbance distributions were shown for sedimentation equilibrium of pfiT 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. Solid lines are the global best-fit distributions using a reversible monomer and dimer model with SEDPHAT . (C) Sedimentation equilibrium analysis of pfiT binding to the HLA-DR1/HA complex. Absorbance distributions were shown for sedimentation equilibrium of the pfiT-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).

Figure 6. The binding site on HLA-DR for pfiT overlaps with that for MAM.

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

Figure 7. Crystal structure of pfiT.

(A) Cartoon representation of crystal structure of native pfiT monomer. Secondary structural elements were labeled. (B) Cartoon representation of crystal structure of the pfiT dimer of the Se-Met pfiT crystal. The N-terminal α1 helix that is missing in the native pfiT structure and dimer interface helices were labeled. (C) Comparison of the dimer of Se-Met pfiT (green and red) with that of native pfiT (blue) reconstituted through crystallographic symmetry in the C2 crystal form. (D) Superposition of pfiT (red) to a putative TetR repressor (blue) from Vibrio parahaemolyticus (PDB code: 3HE0). (E) Superposition of pfiT dimer to that of the QacR-DNA complex (PDB: 1JT0) . pfiT was colored as red and green for the two monomers of the dimer; the QacR dimer was colored yellow. (F) Structure comparison of pfiT with QacR at the QacR DNA-binding site. Residues that are important for DNA-binding were labeled in black (QacR) and green (pfiT), and presented in stick representation, with oxygen in red, nitrogen in blue, and carbon either in green (pfiT) or in yellow (QacR). (G) The electrostatic surface potential of pfiT at the putative DNA-binding site, with blue and red regions indicating positive and negative electrostatic regions, respectively. This figure was made with GRASP . (H) Structure-based alignment of sequences of pfiT and TetR members (TetR , QacR , DesT [33]) 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).