title: Role of the C-terminus in the activity, conformation, and stability of interleukin-6.
abstract: Two murine interleukin-6 (mIL-6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183-187) at the C-terminus (pMC5) and another with the last five residues of mIL-6 substituted by the corresponding residues of human IL-6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was < 0.05% of mIL-6, whereas pMC5H and mIL-6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL-6. Both pMC5 and pMC5H, like mIL-6, failed to interact with recombinant soluble human IL-6 receptor when assayed by surface plasmon resonance-based biosensor analysis. These studies suggest that the C-terminal seven amino acids of human IL-6, alone, do not define species specificity for receptor binding. A variety of biophysical techniques, as well as the binding of a conformational-specific monoclonal antibody, indicated that the global fold of the mIL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr-22 were influenced by the C-terminal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL-6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C-terminal amino acids of IL-6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.
authors.affiliation: Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research, Parkville, Victoria, Australia.
authors.forename: L D
authors.initials: LD
authors.lastname: Ward
authors.forename: A
authors.initials: A
authors.lastname: Hammacher
authors.forename: J G
authors.initials: JG
authors.lastname: Zhang
authors.forename: J
authors.initials: J
authors.lastname: Weinstock
authors.forename: K
authors.initials: K
authors.lastname: Yasukawa
authors.forename: C J
authors.initials: CJ
authors.lastname: Morton
authors.forename: R S
authors.initials: RS
authors.lastname: Norton
authors.forename: R J
authors.initials: RJ
authors.lastname: Simpson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001483:Base Sequence; D004355:Drug Stability; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D015850:Interleukin-6 / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D018123:Receptors, Interleukin / Q000378:metabolism; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D000911:Antibodies, Monoclonal; D015850:Interleukin-6; D010446:Peptide Fragments; D018123:Receptors, Interleukin; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins