Department of Physical Chemistry I, Ruhr University Bochum, Universitätsstrasse 150, 44780 Bochum, Germany.
Biochemistry. 2011 Dec 27;50(51):10990-1000. doi: 10.1021/bi201110h. Epub 2011 Dec 2.
The serine/threonine mammalian sterile 20-like kinase (MST1) is involved in promotion of caspase-dependent and independent apoptosis. Phosphorylation and oligomerization are required for its activation. The oligomerization domain, denoted as SARAH domain, forms an antiparallel coiled coil dimer, and it is important for both MST1 autophosphorylation and interactions with other proteins like the Rassf proteins containing also a SARAH domain. Here we show that the monomeric state of SARAH is thermodynamically unstable and that homodimerization is coupled with folding. Moreover, the influence of the inhibitory domain on SARAH stability and affinity is addressed. By investigating the thermal denaturation using differential scanning calorimetry and circular dichroism, we have found that the SARAH domain dissociates and unfolds cooperatively, without a stable intermediate monomeric state. Combining the data with information from isothermal titration calorimetry, a low thermodynamic stability of the monomeric species is obtained. Thus, it is proposed that the transition from MST1 SARAH homodimer to some specific heterodimer implies a non-native monomer intermediate. The inhibitory domain is found to be highly flexible and intrinsically unfolded, not only in isolation but also in the dimeric state of the inhibitory-SARAH construct. The existence of two caspase recognition motifs within the inhibitory domain suggests that its structural flexibility might be important for activation of MST1 during apoptosis. Moreover, the inhibitory domain increases the thermodynamic stability of the SARAH dimer and the homodimer affinity, while having almost no effect on the SARAH domain in the monomeric state. These results emphasize the importance of flexibility and binding-induced folding for specificity, affinity, and the capacity to switch from one state to another.
丝氨酸/苏氨酸哺乳动物无活性 20 样激酶（MST1）参与促进半胱天冬酶依赖性和非依赖性细胞凋亡。其激活需要磷酸化和寡聚化。寡聚化结构域，称为 SARAH 结构域，形成反平行的卷曲螺旋二聚体，对于 MST1 自身磷酸化以及与其他蛋白（如含有 SARAH 结构域的 Rassf 蛋白）的相互作用都很重要。在这里，我们表明 SARAH 的单体状态热力学上不稳定，同源二聚化与折叠偶联。此外，还探讨了抑制结构域对 SARAH 稳定性和亲和力的影响。通过使用差示扫描量热法和圆二色性研究热变性，我们发现 SARAH 结构域解离并协同展开，没有稳定的中间单体状态。将数据与等温滴定量热法的信息相结合，获得了单体物种的低热力学稳定性。因此，提出 MST1 SARAH 同源二聚体向某些特定异源二聚体的转变意味着存在非天然单体中间物。发现抑制结构域不仅在分离状态下，而且在抑制-SARAH 结构的二聚体状态下，都具有高度的灵活性和固有无序性。抑制结构域内存在两个半胱天冬酶识别基序，表明其结构灵活性对于细胞凋亡过程中 MST1 的激活可能很重要。此外，抑制结构域增加了 SARAH 二聚体的热力学稳定性和同源二聚体的亲和力，而对单体状态下的 SARAH 结构域几乎没有影响。这些结果强调了灵活性和结合诱导折叠对于特异性、亲和力以及从一种状态切换到另一种状态的能力的重要性。
