PDF(Pubmed)
Abstract：
Kinesin-1 is a microtubule motor that transports cellular cargo along microtubules. KIF5A is one of three kinesin-1 isoforms in humans, all of which are autoinhibited by an interaction between the motor and an IAK motif in the proximal region of the C-terminal tail. The C-terminal tail of KIF5A is ∼80 residues longer than the other two kinesin-1 isoforms (KIF5B and KIF5C) and it is unclear if it contributes to autoinhibition. Mutations in KIF5A cause neuronal diseases and could affect autoinhibition, as reported for a mutation that skips exon 27, altering its C-terminal sequence. Here, we combined negative-stain electron microscopy, crosslinking mass spectrometry (XL-MS) and AlphaFold2 structure prediction to determine the molecular architecture of the full-length autoinhibited KIF5A homodimer, in the absence of light chains. We show that KIF5A forms a compact, bent conformation, through a bend between coiled-coils 2 and 3, around P687. XL-MS of WT KIF5A revealed extensive interactions between residues in the motor, between coiled-coil 1 and the motor, between coiled-coils 1 and 2, with coiled-coils 3 and 4, and the proximal region of the C-terminal tail and the motor in the autoinhibited state, but not between the distal C-terminal region and the rest of the molecule. While negative-stain electron microscopy of exon-27 KIF5A splice mutant showed the presence of autoinhibited molecules, XL-MS analysis suggested that its autoinhibited state is more labile. Our model offers a conceptual framework for understanding how mutations within the motor and stalk domain may affect motor activity.
摘要：
Kinesin-1是一种微管马达，可沿着微管运输细胞货物。KIF5A是人类三种驱动蛋白-1亚型之一，所有这些都被电机和C末端尾部近端区域的IAK基序之间的相互作用自动抑制。KIF5A的C末端尾部比其他两种驱动蛋白-1同工型（KIF5B和KIF5C）长80个残基，目前尚不清楚它是否有助于自身抑制。KIF5A的突变会引起神经元疾病，并可能影响自身抑制，如报道的，跳过外显子27的突变，改变其C端序列。这里,我们结合了负染色电子显微镜，交联质谱（XL-MS）和AlphaFold2结构预测，以确定全长自抑制KIF5A同二聚体的分子结构，在没有轻链的情况下。我们证明KIF5A形成了一个紧凑的，弯曲的构象，通过盘绕线圈2和3之间的弯曲，围绕P687。WTKIF5A的XL-MS揭示了电机中残基之间的广泛相互作用，在线圈1和电机之间，在卷曲线圈1和2之间，具有卷曲线圈3和4，以及C末端尾部的近端区域和处于自动禁止状态的电机，但不在远端C末端区域和分子的其余部分之间。虽然外显子-27KIF5A剪接突变体的阴性染色电子显微镜显示存在自抑制分子，XL-MS分析表明其自抑制状态更不稳定。我们的模型提供了一个概念框架，用于了解运动和茎域内的突变如何影响运动活动。
