ROLE OF THE C-TERMINUS IN THE ACTIVITY, CONFORMATION, AND STABILITY OF INTERLEUKIN-6

Two murine interleukin-6 (mIL-6) variants were constructed using the p olymerase chain reaction (PCR), one lacking the last five residues (18 3-187) at the C-terminus (pMC5) and another with the last five residue s of mIL-6 substituted by the corresponding residues of human IL-6 (pM C5H). The growth stimulatory activity of pMC5 on the mouse hybridoma c ell line 7TD1 was <0.05% of mIL-6, whereas pMC5H and mIL-6 were equipo tent. The loss of biological activity of pMC5 correlated with its negl igible receptor binding affinity on 7TD1 cells, while the binding of p MC5H 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 s tudies suggest that the C-terminal seven amino acids of human IL-6, al one, 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 m IL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these c hanges involved residues widely separated in the primary structure. Fo r instance, interactions involving Tyr-22 were influenced by the C-ter minal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity. Equilibrium unfolding experiments indicated that pMC 5 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 muta tion of this region could lead to small but significant alterations in other regions of the molecule.