Supporting Information

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Supporting Figure 4
Supporting Figure 5
Supporting Text
Supporting Figure 6
Supporting Figure 7

Fig. 4. Equilibrium binding analysis of single-site variants. Equilibrium and/or kinetic binding analysis of EP-8 (A) and the T30H (B), E51Q (C), S52aF (D), K53N (E), and E61V (F) mutants interacting with TSST-1 for which SPR sensorgrams, after correction for nonspecific binding, are shown. (Insets in A-E) Nonlinear steady-state affinity analysis for the corresponding interaction. Global fitting of the data to a 1:1 binding model is shown in D-F in black, and the corresponding residual values are plotted below the individual sensorgrams.

Fig. 5. A second hot region on TSST-1 for hVb2.1 interaction. (A-C) Equilibrium analysis of the TSST-1 mutants R68A (A), K71A (B), and S72A (C) binding to EP-8. (Inset in B) Nonlinear steady-state affinity analysis for the corresponding interaction. (D-F) Representative flow cytometry plots for Vb2-specific activation of peripheral blood lymphocytes by TSST-1 proteins R68A (D), K71A (E) , and S72A (F). Plots are a representative experiment from a single donor. (G) Quantitation of activated (CD25⁺) or nonactivated (CD25^-) Vb2⁺CD3⁺ T cells. Data shown are the average ± SEM from three independent experiments for WT TSST-1, R68A, K71A, and S72A. Q139A was previously shown to lack binding to soluble Vb2.1 and did not activate primary human T cells [McCormick, J. K., Tripp, T. J., Llera, A. S., Sundberg, E. J., Dinges, M. M., Mariuzza, R. A. & Schlievert, P. M. (2003) J. Immunol. 171, 1385-1392].

Fig. 6. Kinetic analysis of multisite variants. SPR sensorgrams, after correction for nonspecific binding, for the E51Q/K53N (A), E51Q/K53N/E61V (B), D10 (C), and S52aF/K53N/E61V (D) mutants binding to TSST-1 are shown. Global fitting of the data to a 1:1 binding model is shown in black, and the corresponding residual values are plotted below the individual sensorgrams. (Inset in A) Nonlinear steady-state affinity analysis for the corresponding interaction.

Fig. 7. Hydrophobic mutations at position 61 maintain inter-hot regional cooperativity. Kinetic analysis of the E61I (A and B), E61L (C and D), E61F (E and F), and E61W (G and H) mutations in the WT (A, C, E, and G) and E51Q/K53N (B, D, F, and H) backgrounds. Global fitting of the data to a 1:1 binding model is shown in black, and the corresponding residual values are plotted below the individual sensorgrams.

Supporting Text

Activation of Primary Human Vb2⁺ T Cells by TSST-1 Proteins. Peripheral blood mononuclear cells (1 × 10⁶ per ml) from three healthy human donors were suspended in R10 medium [RPMI medium 1640 (GIBCO/Invitrogen) supplemented with 10% FCS (Sigma), 100 mg/ml streptomycin (HyClone), 100 units/ml penicillin (HyClone), 2 mM L-glutamine (HyClone), 1 mM MEM sodium pyruvate (HyClone), 100 mM nonessential amino acid (HyClone), and 25 mM Hepes, pH 7.0 (BioShop, Burlington, ON, Canada)]. Cells were activated with 1 ng/ml various TSST-1 proteins for 4 days, and expression of CD3, Vb2, and CD25 was analyzed in a FACSCalibur flow cytometer (BD Biosciences). The mAbs used were as follows: FITC-labeled anti-Vb2 (Immunotech, Beckman Coulter), phycoerythrin-labeled anti-CD25 (BD Bioscience Pharmingen), and PC5 labeled anti-CD3 (Immunotech, Beckman Coulter). Data analysis was performed with FLOWJO software.