Definition of receptor binding sites on human interleukin-11 by molecular modeling-guided mutagenesis.
Interleukin-11 (IL-11) belongs to the interleukin-6 (IL-6)-type subfamily of long-chain helical cytokines including IL-6, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M, and cardiotrophin-1, which all share the glycoprotein gp130 as a signal transducing receptor component. IL-11 acts on cells expressing gp130 and the IL-11 receptor (IL-11R) alpha-subunit (IL-11Ralpha). The structural epitopes of IL-11 required for the recruitment of the individual receptor subunits have not yet been defined. Based on the structure of CNTF, a three-dimensional model of human IL-11 was built. Using this model, 10 surface exposed amino acid residues of IL-11 were selected for mutagenesis using analogies to the well-characterized receptor recruitment sites of IL-6, CNTF, and LIF. The respective mutants of human IL-11 were expressed as soluble fusion proteins in bacteria. Their biological activities were determined on HepG2 and Ba/F3-130-11alpha cells. Several mutants with substantially decreased bioactivity and one hyperagonistic mutant were identified and further analyzed with regard to recruitment of IL-11Ralpha and gp130. The low-activity mutant I171D still binds IL-11Ralpha but fails to recruit gp130, whereas the hyperagonistic variant R135E more efficiently engages the IL-11R subunits. The low-activity mutants R190E and L194D failed to bind to IL-11Ralpha. These findings reveal a common mechanism of receptor recruitment in the family of IL-6-type cytokines and offer considerable perspectives for the rational design of IL-11 antagonists and hyperagonists.
Tacken
I
I
Institut für Biochemie, Rheinisch-Westfälische Technische Hochschule Aachen, Germany.
Dahmen
H
H
Boisteau
O
O
Minvielle
S
S
Jacques
Y
Y
Grötzinger
J
J
Küster
A
A
Horsten
U
U
Blanc
C
C
Montero-Julian
F A
FA
Heinrich
P C
PC
Müller-Newen
G
G
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000378:metabolism*; D001665:Binding Sites; D002455:Cell Division; D050822:Cytokine Receptor gp130; D016207:Cytokines / Q000819:agonists / Q000037:antagonists & inhibitors; D006131:Growth Inhibitors; D006801:Humans; D017370:Interleukin-11 / Q000737:chemistry / Q000235:genetics*; D015850:Interleukin-6 / Q000378:metabolism; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D008222:Lymphokines; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D018121:Receptors, Cytokine / Q000378:metabolism; D053606:Receptors, OSM-LIF; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013879:Thioredoxins / Q000235:genetics; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D016207:Cytokines; D006131:Growth Inhibitors; C494199:IL6ST protein, human; D017370:Interleukin-11; D015850:Interleukin-6; C507837:LIF protein, human; C507840:LIFR protein, human; D053605:Leukemia Inhibitory Factor; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D008222:Lymphokines; D008562:Membrane Glycoproteins; D018121:Receptors, Cytokine; D053606:Receptors, OSM-LIF; D011993:Recombinant Fusion Proteins; D050822:Cytokine Receptor gp130; D013879:Thioredoxins