Homodimeric cross-over structure of the human granulocyte colony-stimulating factor (GCSF) receptor signaling complex

Abstract

A crystal structure of the signaling complex between human granulocyte colony-stimulating factor (GCSF) and a ligand binding region of GCSF receptor (GCSF-R), has been determined to 2.8 A resolution. The GCSF:GCSF-R complex formed a 2:2 stoichiometry by means of a cross-over interaction between the Ig-like domains of GCSF-R and GCSF. The conformation of the complex is quite different from that between human GCSF and the cytokine receptor homologous domain of mouse GCSF-R, but similar to that of the IL-6/gp130 signaling complex. The Ig-like domain cross-over structure necessary for GCSF-R activation is consistent with previously reported thermodynamic and mutational analyses.

Fig. 1.

Overall structure of the 2:2 complex between hGCSF and hGCSF-R (Ig–CRH domain). The GCSF molecules are colored in red and orange, and GCSF-R molecules are colored in green and cyan. Fig. 1 was prepared by the pymol Molecular Graphics System (DeLano Scientific, San Carlos, CA).

Fig. 2.

Amino acid sequence alignments between ligands (hGCSF, hIL-6, and vIL-6) (a) and Ig-CRH domains of human receptors (GCSF-R and gp130) (b). The sequence identity between GCSF and IL-6 is 18%, and the identity between the Ig–CRH domains of hGCSF-R and human gp130 is 29%. The secondary structure elements [helices and strands; calculated by procheck (35)] are also shown as wavy (cyan) and solid (purple) lines, respectively. Identical amino acid residues are shaded in black. The residues concerned with ligand–receptor binding at sites I, II, and III are colored by green, red, and blue, respectively. Asterisks and pluses show binding sites at site II of gp130 in hIL-6 and vIL-6 complex, respectively. Structurally disordered regions are shown as gray. Three free cysteines, which are colored yellow, in Ig–CRH domains of GCSF-R are mutated to serines in this study.

Fig. 3.

Close-up view of the interface between ligand and receptor. (a) Site II. (b) Site III. Related residues of ligand–receptor binding are drawn as stick model. Figs. 3–5 were prepared by weblab viewer lite (Accelrys Inc., San Diego).

Fig. 4.

The 2:2 conformation between ligand and receptor. (a) hGCSF and hGCSF-R (Ig–CRH domain). (b) hGCSF and mouse GCSF-R (CRH domain). (c) hIL-6, human gp130 (Ig–CRH domain), and hIL-6Rα. (d) vIL-6 and human gp130. Ligands are colored in red and orange. Receptors are colored in green and cyan. The IL-6Rα molecules are drawn by wire model. Dashed lines and ellipse show twofold axis.

Fig. 5.

Structural comparison of GCSF-R complex and gp130 complex. (a) Site II. (b) Site III. Complexes are superimposed in a and separated in b. The GCSF, GCSF-R, IL-6, and gp130 molecules are colored in red, green, cyan, and orange, respectively. Related residues of specificity are drawn by stick model.