Crystallization and preliminary X-ray diffraction of human interleukin-7 bound to unglycosylated and glycosylated forms of its alpha-receptor

Abstract

The interleukin-7 (IL-7) signaling pathway plays an essential role in the development, proliferation and homeostasis of T and B cells in cell-mediated immunity. Understimulation and overstimulation of the IL-7 signaling pathway leads to severe combined immunodeficiency, autoimmune reactions, heart disease and cancers. Stimulation of the IL-7 pathway begins with IL-7 binding to its alpha-receptor, IL-7R alpha. Protein crystals of unglycosylated and glycosylated complexes of human IL-7-IL-7R alpha extracellular domain (ECD) obtained using a surface entropy-reduction approach diffract to 2.7 and 3.0 A, respectively. Anomalous dispersion methods will be used to solve the unglycosylated IL-7-IL-7R alpha ECD complex structure and this unglycosylated structure will then serve as a model in molecular-replacement attempts to solve the structure of the glycosylated IL-7-alpha-receptor complex.

Figure 1

Analytical size-exclusion chromatography experiments on wt IL-7 (EC) and IL-­7Rα (EC) ECD. A Superdex 200 10/300 GL SEC column (GE Healthcare) was equilibrated with PBS buffer pH 7.4 at a flow rate of 0.5 ml min⁻¹ at 277 K. The blue chromatograph is an injection of 10 µM IL-7 (EC) with a retention time of 33.7 min. The red chromatograph is an injection of 5 µM IL-7Rα (EC) running at 31.8 min. The black chromatograph is an injection of a 2:1 molar ratio of IL-7 (EC):IL-7Rα (EC) with retention times of 29.8 and 33.6 min. The arrows above the chromatographs indicate the molecular weights of known standards.

Figure 2

Crystals of the 1:1 complexes of E106A-IL-7 with unglycosylated and glycosylated IL-7Rα ECD. (a) Small rod-shaped protein crystals of the unglycosylated E106A-IL-7–IL-7Rα (EC) ECD complex with dimensions of <40 µm in all dimensions. (b) Prism-shaped protein crystal of the complex of E106A-IL-7 with glycosylated IL-­7Rα (S2) ECD with dimensions of approximately of 50 × 40 × 10 µm. (c) Native X-ray diffraction image of a unglycosylated E106A-IL-7–IL-7Rα (EC) ECD complex crystal with diffraction to 2.7 Å.