Löw Christian, Homeyer Nadine, Weininger Ulrich, Sticht Heinrich, Balbach Jochen
Institut für Physik, Biophysik, Martin-Luther-Universität Halle-Wittenberg, D-06120 Halle (Saale), Germany.
ACS Chem Biol. 2009 Jan 16;4(1):53-63. doi: 10.1021/cb800219m.
P19INK4d consists of five ankyrin repeats and controls the human cell cycle by inhibiting the cyclin D-dependent kinases 4 and 6. Posttranslational phosphorylation of p19INK4d has been described for Ser66 and Ser76. In the present study we show that mimicking the phosphorylation site of p19INK4d by a glutamate substitution at position 76 dramatically decreases the stability of the native but not an intermediate state. At body temperature the native conformation is completely lost and p19INK4d molecules exhibit the intermediate state as judged by kinetic and equilibrium analysis. High resolution NMR spectroscopy verified that the three C-terminal repeats remained folded in the intermediate state, whereas all cross-peaks of the two N-terminal repeats lost their native chemical shift. Molecular dynamic simulations of p19INK4d in different phosphorylation states revealed large-scale motions in phosphorylated p19INK4d, which cause destabilization of the interface between the second and third ankyrin repeat. Only doubly phosphorylated p19INK4d mimic mutants showed in vitro an increased accessibility for ubiquitination, which might be the signal for degradation in vivo.
p19INK4d由五个锚蛋白重复序列组成，通过抑制细胞周期蛋白D依赖性激酶4和6来控制人类细胞周期。p19INK4d的Ser66和Ser76位点存在翻译后磷酸化现象。在本研究中，我们发现，通过在第76位用谷氨酸替代来模拟p19INK4d的磷酸化位点，会显著降低天然状态而非中间状态的稳定性。在体温下，天然构象完全丧失，根据动力学和平衡分析判断，p19INK4d分子呈现中间状态。高分辨率核磁共振光谱证实，三个C末端重复序列在中间状态下仍保持折叠，而两个N末端重复序列的所有交叉峰均失去了其天然化学位移。不同磷酸化状态下p19INK4d的分子动力学模拟显示，磷酸化的p19INK4d存在大规模运动，这导致第二个和第三个锚蛋白重复序列之间的界面不稳定。只有双磷酸化的p19INK4d模拟突变体在体外显示出泛素化的可及性增加，这可能是体内降解的信号。
