Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore, Singapore.
PLoS One. 2011;6(11):e27072. doi: 10.1371/journal.pone.0027072. Epub 2011 Nov 1.
T46I is the second mutation on the hVAPB MSP domain which was recently identified from non-Brazilian kindred to cause a familial amyotrophic lateral sclerosis (ALS). Here using CD, NMR and molecular dynamics (MD) simulations, we characterized the structure, stability, dynamics and binding capacity of the T46I-MSP domain. The results reveal: 1) unlike P56S which we previously showed to completely eliminate the native MSP structure, T46I leads to no significant disruption of the native secondary and tertiary structures, as evidenced from its far-UV CD spectrum, as well as Cα and Cβ NMR chemical shifts. 2) Nevertheless, T46I does result in a reduced thermodynamic stability and loss of the cooperative urea-unfolding transition. As such, the T46I-MSP domain is more prone to aggregation than WT at high protein concentrations and temperatures in vitro, which may become more severe in the crowded cellular environments. 3) T46I only causes a 3-fold affinity reduction to the Nir2 peptide, but a significant elimination of its binding to EphA4. 4) EphA4 and Nir2 peptide appear to have overlapped binding interfaces on the MSP domain, which strongly implies that two signaling networks may have a functional interplay in vivo. 5) As explored by both H/D exchange and MD simulations, the MSP domain is very dynamic, with most loop residues and many residues on secondary structures highly fluctuated or/and exposed to bulk solvent. Although T46I does not alter overall dynamics, it does trigger increased dynamics of several local regions of the MSP domain which are implicated in binding to EphA4 and Nir2 peptide. Our study provides the structural and dynamic understanding of the T46I-causing ALS; and strongly highlights the possibility that the interplay of two signaling networks mediated by the FFAT-containing proteins and Eph receptors may play a key role in ALS pathogenesis.
T46I 是 hVAPB MSP 结构域的第二个突变，最近在非巴西家族中发现该突变可导致家族性肌萎缩侧索硬化症（ALS）。在这里，我们使用圆二色性（CD）、核磁共振（NMR）和分子动力学（MD）模拟，对 T46I-MSP 结构域的结构、稳定性、动力学和结合能力进行了表征。结果表明：1）与我们之前证明完全消除天然 MSP 结构的 P56S 不同，T46I 并没有导致天然二级和三级结构的显著破坏，这可以从其远紫外 CD 光谱以及 Cα 和 Cβ NMR 化学位移得到证明。2）然而，T46I 确实导致热力学稳定性降低和协同脲解折叠转变的丧失。因此，与 WT 相比，T46I-MSP 结构域在高蛋白浓度和体外高温下更容易聚集，在拥挤的细胞环境中可能会更加严重。3）T46I 仅导致 Nir2 肽的亲和力降低 3 倍，但对 EphA4 的结合能力显著丧失。4）EphA4 和 Nir2 肽似乎在 MSP 结构域上具有重叠的结合界面，这强烈表明两种信号网络在体内可能具有功能相互作用。5）通过 H/D 交换和 MD 模拟探索，MSP 结构域非常动态，大多数环残基和许多二级结构上的残基高度波动或/和暴露于溶剂中。尽管 T46I 不会改变整体动力学，但它确实会引发 MSP 结构域的几个局部区域的动力学增加，这些区域与 EphA4 和 Nir2 肽的结合有关。我们的研究提供了 T46I 引起 ALS 的结构和动力学理解；并强烈强调了由含有 FFAT 的蛋白质和 Eph 受体介导的两个信号网络的相互作用可能在 ALS 发病机制中起关键作用的可能性。
