NMR studies of human interleukin-6 and its mutants

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On the basis of the partial signal assignments and the observed NOE network, the folding topology of human IL-6 was analyzed. A comparison of the folding topology of IL-6 with that of human G-CSF indicated that IL-6 has a significant similarity of folding topology to that of G-CSF.DSC thermogram of the wild-type IL-6 at pH 4.2 showed two endothermic peaks at 35 and 65ﾟC,indicating that an intermediate state exists in the heat-denaturation pathway. In order to understand the structure-function and structure-stability relationships in the human IL-6 system, comparative studies were performed on the basis of NMR,DSC,and CD data obtained using the wild-type IL-6 and six mutants (L152V,L159V,L166V,L168V,L175V,and L182V). The NMR data showed that L182V substitution induced no structural change in IL-6, suggesting that Leu182 is located on the surface of the IL-6 molecule. A significant decrease in receptor-binding activity was observed in the L182V mutant. It was concluded that the side-chai … More n of Leu182 is directly involved in receptor-binding. L175V substitution was shown to induce a significant structural change in IL-6. It is possible that helix D bent more sharply toward helix B in the L175V mutant than in the wild-type IL-6 to maintain a closely packed and solvent-inaccessible core formed in the mutated region. It is suggested that the kink of helix D is related to the decrease in receptor-binding activity in the L175V mutant. In the case of L152V mutant, a significant structural changes were observed compared with the wild-type IL-6. However, no difference in receptor-binding activity between the wild-type IL-6 and L152V mutant was observed. The DSC data revealed that the partial unfolding of L152V mutant and the full unfolding of L175V mutant occurred at temperatures lower than those of the wild-type IL-6. The NMR data observed at various temperatures showed that L152V and L175V mutants are prone to the soluble self-association and insoluble precipitation, respectively, compared with the wild-type IL-6. Less