NMR studies of human interleukin-6 and its mutants

基于部分信号分配和观察到的NOE网络，分析了人IL-6的折叠拓扑结构。将IL-6的折叠拓扑结构与人G-CSF的折叠拓扑结构进行比较，发现IL-6与G-CSF的折叠拓扑结构具有显著的相似性。野生型IL-6在pH 4.2时的DSC热谱图在35和65℃处显示出两个吸热峰，表明热变性途径存在中间状态。为了了解人类IL-6系统的结构-功能和结构-稳定性关系，我们利用野生型IL-6和6个突变体（L152V、L159V、L166V、L168V、L175V和L182V）的NMR、DSC和CD数据进行了比较研究。核磁共振数据显示，L182V取代没有引起IL-6的结构变化，表明Leu182位于IL-6分子的表面。在L182V突变体中观察到受体结合活性显著降低。结果表明，Leu182的side-chai - More n直接参与了受体结合。L175V取代可诱导IL-6发生显著的结构变化。与野生型IL-6相比，在L175V突变体中，螺旋D向螺旋B弯曲的程度可能更大，以维持在突变区域形成的紧密排列和溶剂不可接近的核心。这表明，螺旋D的扭结与L175V突变体中受体结合活性的降低有关。在L152V突变体中，与野生型IL-6相比，观察到显著的结构变化。然而，野生型IL-6和L152V突变体之间的受体结合活性没有差异。DSC数据显示，与野生型IL-6相比，L152V突变体的部分展开和L175V突变体的完全展开发生在较低的温度下。在不同温度下观察到的核磁共振数据表明，与野生型相比，L152V和L175V突变体分别倾向于可溶性自结合和不溶性沉淀。少

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