1H, 13C and 15N resonance assignments of a highly-soluble murine interleukin-3 analogue with wild-type bioactivity

Abstract

Interleukin-3 (IL-3) is a cytokine that acts as a critical mediator of inflammation and immune responses to infections. IL-3, like interleukin-5 (IL-5) and granulocyte-macrophage colony stimulating factor (GM-CSF), exerts its effects on target cells via receptors composed of cytokine-specific alpha-subunits and a common beta-subunit (betac-subunit, shared with IL-5 and GM-CSF). In contrast to humans, mice also possess an additional beta-receptor, beta(IL-3), that can specifically bind IL-3. Except for a study carried out on an analogue of human IL-3 that contains 14 mutations, structure-related studies of IL-3 have been very limited, largely because of its poor solution behaviour. Here we report (1)H, (13)C, and (15)N chemical shift assignments of murine IL-3 comprising residues 33-156 (SWISS-PROT accession number: P01586), in which the only mutation is an alanine substitution of Cys105. The mIL-3 construct used in the present study was engineered by eliminating residues 27-32 of the N-terminus (the first 26 residues of the primary sequence of mIL-3 are cleaved in vivo during secretion), the C-terminal 10 residues (157-166), and a disulfide bond between Cys105 and Cys166 that is poorly conserved in orthologue sequences. The new construct vastly improves the solubility of murine IL-3 while maintaining its wild-type biological activity.

Murine Interleukin-3: Structure, Dynamics, and Conformational Heterogeneity in Solution

“…The optimal solution conditions were established to be 20 mM potassium phosphate, pH 6.7, and 283 K. Under these conditions, near-complete assignments of 1 H, 15 N, and 13 C chemical shifts were obtained using standard triple-resonance heteronuclear experiments. 18 A total of 1209 nonredundant distance restraints obtained from 15 N-and 13 C-edited NOESY spectra, together with 180 dihedral angle restraints derived from secondary chemical shifts and 28 hydrogen bond restraints (all within individual helices), were used in calculating the 3D structure of mIL-3 . The statistics for the 20 lowest energy structural models (Table 2) indicate that they fit well with experimentally derived distance and angle restraints.…”

“…The expression and purification of mIL-3 33-156 used for the present studies have been described previously. 11,18 NMR samples were prepared by dissolving lyophilized 15 N-labeled (ca. 0.5, 1.0, or 2.0 mg) or 13 C/ 15 N-double-labeled (ca.…”

“…Chemical shift assignments of mIL-3 33-156 have been deposited in BioMagResBank (http://www.bmrb.wisc.edu, with accession code 16614). 18 Distance restraints were derived from the intensities of NOE cross-peaks presented in 15 N-edited and 13 Cedited 3D NOESY-HSQC spectra (both with a mixing time of 120 ms) recorded using a Bruker Avance 800 spectrometer equipped with a cryoprobe. Backbone dihedral angle (φ and ψ) restraints were obtained from assigned chemical shifts using the program TALOS.…”

Applications of nuclear shielding

“…: P01586) were reported, in which the only mutation is an alanine substitution of Cys105. 309 Sticholysin II is an actinoporin of 175 amino acids produced by the sea anemone Stichodactyla helianthus. As a first step towards a 3D structural characterisation and its interaction with membrane models at a residue level, herein, the nearly complete NMR 15 N, 13 C and 1 H chemical shifts assignments of the Y111N variant at pH 4.0 and 25 1C (BMRB No.…”