Figure 2. The acidic pocket is required for the Be²⁺-dependent functional and biophysical changes in DP2-peptide complexes.

(A) Equivalent amounts of biotinylated MHCII-peptide complexes (DP2-M2, DP2-M2(NQ), DP2(β69K)-M2, and HLA-DR52c-pHIR) were immobilized in separate culture wells and used to stimulate the AV22 transfectoma T cell with or without various concentrations of BeSO4. IL-2 secretion was assessed at 24 hr. The experiment was repeated with equivalent results.

(B) Biotinylated DP2-M2 (Approximately 2,000 RU) was captured in a flowcell of an SA-biosensor chip in a BIAcore 2000 instrument. Various concentrations of soluble AV22 TCR were injected for 140s through the flowcell, before and after loading of the immobilized DP2-M2 with Be²⁺ . The surface plasmon resonance signal (RU) was followed both during and after the injection and the binding kinetics analyzed with BIAcore BIAevaluation 4.1 software. The experiment was repeated four times with similar results.

(C) The plateau equilibrium RU data in (B) were used to create a Scatchard plot. The dissociation constant (KD) and regression coefficient (R) were calculated from the linear least-squares fit to the data.

(D) Same as (B) except that biotinylated DP2-M2(NQ) and DP2(β69K)-M2 were immobilized in separate flowcells. Only data for the injection with 5 µM AV22 TCR is shown. The experiment was repeated with similar results.

(E) The thermal stability of the series of soluble MHCII-peptide complexes shown was assessed in the presence or absence of Be2+ using differential scanning fluorimetry with the fluorescent dye SYPRO orange. The melting temperature (Tm) was defined as the temperature needed to achieve the half maximal increase in fluorescence. Results are pooled and averaged from six experiments. Each melting curve was performed at least three times among the experiments.