Zinc induces dimerization of the class II major histocompatibility complex molecule that leads to cooperative binding to a superantigen

Abstract

Dimerization of class II major histocompatibility complex (MHC) plays an important role in the MHC biological function. Mycoplasma arthritidis-derived mitogen (MAM) is a superantigen that can activate large fractions of T cells bearing specific T cell receptor Vbeta elements. Here we have used structural, sedimentation, and surface plasmon resonance detection approaches to investigate the molecular interactions between MAM and the class II MHC molecule HLA-DR1 in the context of a hemagglutinin peptide-(306-318) (HA). Our results revealed that zinc ion can efficiently induce the dimerization of the HLA-DR1/HA complex. Because the crystal structure of the MAM/HLA-DR1/hemagglutinin complex in the presence of EDTA is nearly identical to the structure of the complex crystallized in the presence of zinc ion, Zn(2+) is evidently not directly involved in the binding between MAM and HLA-DR1. Sedimentation and surface plasmon resonance studies further revealed that MAM binds the HLA-DR1/HA complex with high affinity in a 1:1 stoichiometry, in the absence of Zn(2+). However, in the presence of Zn(2+), a dimerized MAM/HLA-DR1/HA complex can arise through the Zn(2+)-induced DR1 dimer. In the presence of Zn(2+), cooperative binding of MAM to the DR1 dimer was also observed.

FIGURE 1

Structural comparison of the MAM/HLA-DR1/HA complex crystallized under different conditions as follows: 1 mM Zn(OAc)₂ (black); no Zn²⁺, EDTA, or Cd²⁺ (light gray).

FIGURE 2. Sedimentation-velocity analysis of the binding of MAM to HLA-DR1/HA

A, sedimentation coefficient distributions of MAM alone (△), of DR1 alone (○), and of mixtures at different ratio of concentrations as follows: MAM:DR1 = 6:6 μM (□); = 30:10 μM (■); and = 5:10 μM (●) in the absence of Zn²⁺. B, sedimentation coefficient distributions of DR1 alone and MAM/DR1 mixtures under various conditions. □, 8 μM DR1 with 1 mM EDTA; □, 15 μM DR1 with 1 mM ZnCl₂; ○, 6 μM MAM and 4 μM DR1 with 1 mM EDTA; △, 6 μM MAM and 4 μM DR1 with 1 mM ZnCl₂; ●, 16 μM MAM and 1 μM DR1 with 1 mM ZnCl₂; ◇,16 μM MAM and 1 μM DR1 with 1 mM ZnCl₂ and 10 mM EDTA.

FIGURE 3. Affinity measurement of binding of MAM to the HLA-DR1/HA complex in the absence of Zn²⁺

A, SDS-PAGE analysis of biotinylated HLA-DR1/HA complex. Lane 1, Bio-Rad broad range molecular weight standard; lane 2, biotinylated HLA-DR1/HA complex under boiled condition; lane 3, biotinylated HLA-DR1/HA complex under nonboiled condition. B, BIAcore sensograms of kinetic data for MAM binding to the HLA-DR1/HA complex with the high salt HBS-EP buffer (10 mM Hepes (pH 7.4), 1 M NaCl, 3.4 mM EDTA, 0.05% P-20). Biotinylated HLA-DR1/HA complex was coupled to an SA chip (~260 RU). The concentrations for the injected MAM samples were from 8 μM to 32 nM, with 2-fold dilutions. Global fitting of data to a 1:1 binding model is shown in dark black. C, sedimentation equilibrium analysis of MAM binding to the HLA-DR1/HA complex in the presence of 5 mM EDTA. Representative absorbance distributions for the sedimentation equilibrium of 5 μM MAM mixed with 3 μM HLA-DR1/HA at 20 °C at rotor speeds of 20,000 rpm (□), 25,000 rpm (○), and 30,000 rpm (△). Distributions were analyzed as part of a global fitting to the absorbance data at multiple loading concentrations. Solid lines are the global best fit distributions using a reversible A + B ⇋ AB model.

FIGURE 4. Sedimentation equilibrium analysis of Zn²⁺-induced dimerization of the HLA-DR1/HA complex and the MAM/HLA-DR1/HA complex at rotor speeds of 20,000 (□ or ■), 25,000 rpm (○ or ●), and 30,000 rpm (△ or ▲), unless specified

A, representative absorbance distributions for the sedimentation equilibrium of 15 μM MAM with 1 mM Zn²⁺ (■, ●, and ▲) and without Zn²⁺ (□, ○, and △). B, HLA-DR1/HA complex at 5 μM concentration with 1 mM Zn²⁺ (■, ●, and ▲) and without Zn²⁺ (□, ○, and △), at three rotor speeds of 18,000 (□ or ■), 21,000 rpm (○ or ●), and 24,000 rpm (△ or ▲). C, HLA-DR1/HA complex at 2 μM with ZnCl₂ at concentrations of 0.1 mM (□, ○, and ▲) and 0.3 mM (■, ●, and ▲), respectively. D, the MAM/HLA-DR1/HA complex with 1 mM ZnCl₂ at rotor speeds of 15,000 rpm (○), 20,000 rpm (▲), 25,000 rpm (◇), and 30,000 rpm (□). Distributions were analyzed as part of a global fitting to the absorbance data at multiple loading concentrations. Solid lines are the global best fit distributions.