Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding.
Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 alpha-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability (Ward LD et al., 1993, Protein Sci 2:1472-1481). To further probe the structure-function relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of alpha-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.
Hammacher
A
A
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research/Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia.
Ward
L D
LD
Weinstock
J
J
Treutlein
H
H
Yasukawa
K
K
Simpson
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D015374:Biosensing Techniques; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D002942:Circular Dichroism; D005340:Fibrinogen / Q000096:biosynthesis; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry* / Q000378:metabolism; D008113:Liver Neoplasms / Q000473:pathology; D008213:Lymphocyte Activation; D051379:Mice; D008958:Models, Molecular; D009101:Multiple Myeloma / Q000473:pathology; D011485:Protein Binding; D011487:Protein Conformation; D018123:Receptors, Interleukin / Q000378:metabolism*; D019947:Receptors, Interleukin-6; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D015850:Interleukin-6; D018123:Receptors, Interleukin; D019947:Receptors, Interleukin-6; D011993:Recombinant Fusion Proteins; D016179:Granulocyte Colony-Stimulating Factor; D005340:Fibrinogen