Purification, crystallization and preliminary X-ray diffraction of a complex between IL-10 and soluble IL-10R1

Abstract

A complex between interleukin-10 and the extracellular domain of its high-affinity receptor (sIL-10R1) has been crystallized from polyethylene glycol solutions. Crystals suitable for diffraction analysis required the modification of the NXS/T glycosylation sites on sIL-10R1 by site-directed mutagenesis and inclusion of the detergent cyclohexyl-methyl-beta-D-maltopyranoside in the crystallization experiments. The crystals belong to space group P3(2)12 or its enantimorph P3(1)12, with unit-cell parameters a = b = 46.23, c = 307.78 A, alpha = beta = 90, gamma = 120 degrees, and diffract X-rays to approximately 2.9 A. The IL-10 dimer is positioned on a crystallographic twofold, resulting in one IL-10 chain and one sIL-10R1 chain in the asymmetric unit, which corresponds to a solvent content of approximately 44%.

Crystal structure of human cytomegalovirus IL-10 bound to soluble human IL-10R1

“…sIL-10R1Q6 was expressed and purified as described (25). cmvIL-10 (Townes strain) was secreted into the culture media from Drosophila S2 cells (Invitrogen) and purified by affinity chromatography with sIL-10R1 beads.…”

Epstein-Barr Virus IL-10 Engages IL-10R1 by a Two-step Mechanism Leading to Altered Signaling Properties

“…T213C was expressed in Drosophila S2 cells and purified using an hIL-10 affinity column (25). The receptor was biotinylated with polyethelene oxide-maleimide-activated biotin (Pierce) as described previously (26).…”

Structural Features of the Interleukin-10 Family of Cytokines

“…Unfortunately, IL-10R2 is a low-affinity receptor [9,48], and it is difficult, if not impossible, to obtain a stable ternary complex capable to crystallize. However, the binary intermediate complexes of both hIL-10 and CMVIL-10 with soluble receptor sIL-10R1 are quite stable and both were crystallized [49]. It was also shown that in solution IL-10 and sIL-10R1 form a complex consisting of two IL-10 dimers and four molecules of sIL-10R1 [50,51]; therefore, the question of the exact stoichiometry of the signaling ternary complex is also open.…”

“…Crystals of a complex of hIL-10 [49] and CMVIL-10 with non-glycosylated mutant of the extracellular domain of IL-10R1 were obtained and both crystal structures were solved [25,52] at resolution 2.9 Å and 2.7 Å, respectively. An asymmetric part of the crystal unit cell of the hIL-10 complex is composed of one domain of hIL-10 and one molecule of sIL-10R1.…”