Tripet B, Vale R D, Hodges R S
Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
J Biol Chem. 1997 Apr 4;272(14):8946-56. doi: 10.1074/jbc.272.14.8946.
Kinesin is a dimeric motor protein that can move for several micrometers along a microtubule without dissociating. The two kinesin motor domains are thought to move processively by operating in a hand-over-hand manner, although the mechanism of such cooperativity is unknown. Recently, a approximately 50-amino acid region adjacent to the globular motor domain (termed the neck) has been shown to be sufficient for conferring dimerization and processive movement. Based upon its amino acid sequence, the neck is proposed to dimerize through a coiled-coil interaction. To determine the accuracy of this prediction and to investigate the possible function of the neck region in motor activity, we have prepared a series of synthetic peptides corresponding to different regions of the human kinesin neck (residues 316-383) and analyzed each peptide for its respective secondary structure content and stability. Results of our study show that a peptide containing residues 330-369 displays all of the characteristics of a stable, two-stranded alpha-helical coiled-coil. On the other hand, the NH2-terminal segment of the neck (residues approximately 316-330) has the capacity to adopt a beta-sheet secondary structure. The COOH-terminal residues of the neck region (residues 370-383) are not alpha-helical, nor do they contribute significantly to the overall stability of the coiled-coil, suggesting that these residues mark the beginning of a hinge located between the neck and the extended alpha-helical coiled coil stalk domain. Interestingly, the two central heptads of the coiled-coil segment in the neck contain conserved, "non-ideal" residues located within the hydrophobic core, which we show destabilize the coiled-coil interaction. These residues may enable a portion of the coiled-coil to unwind during the mechanochemical cycle, and we present a model in which such a phenomenon plays an important role in kinesin motility.
驱动蛋白是一种二聚体马达蛋白，能够沿着微管移动数微米而不解离。尽管这种协同作用的机制尚不清楚，但人们认为两个驱动蛋白马达结构域以交替的方式进行连续移动。最近，已证明与球状马达结构域相邻的一个约50个氨基酸的区域（称为颈部）足以实现二聚化和连续移动。根据其氨基酸序列，推测颈部通过卷曲螺旋相互作用实现二聚化。为了确定这一预测的准确性，并研究颈部区域在马达活性中可能的功能，我们制备了一系列对应于人驱动蛋白颈部不同区域（残基316 - 383）的合成肽，并分析了每种肽各自的二级结构含量和稳定性。我们的研究结果表明，包含残基330 - 369的肽展现出稳定的双链α - 螺旋卷曲螺旋的所有特征。另一方面，颈部的NH2末端片段（残基约316 - 330）能够形成β - 折叠二级结构。颈部区域的COOH末端残基（残基370 - 383）不是α - 螺旋结构，也对卷曲螺旋的整体稳定性贡献不大，这表明这些残基标志着位于颈部和延伸的α - 螺旋卷曲螺旋柄结构域之间的铰链的起点。有趣的是，颈部卷曲螺旋片段的两个中央七肽包含位于疏水核心内的保守“非理想”残基，我们发现这些残基会破坏卷曲螺旋相互作用。这些残基可能使卷曲螺旋的一部分在机械化学循环中解旋，我们提出了一个模型，其中这种现象在驱动蛋白的运动中起重要作用。
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