Nucleotide-induced flexibility change in neck linkers of dimeric kinesin as detected by distance Measurements using spin-labeling EPR.
Nucleotide-induced flexibility change in neck linkers of dimeric kinesin as detected by distance Measurements using spin-labeling EPR.
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DOI：
10.1016/j.jmb.2008.12.079
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发表时间：
2009-02
影响因子：
5.6
通讯作者：
K. Sugata;Likai Song;Motoyoshi Nakamura;S. Ueki;P. Fajer;T. Arata
中科院分区：
文献类型：
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作者：
K. Sugata;Likai Song;Motoyoshi Nakamura;S. Ueki;P. Fajer;T. Arata
利用偶极连续波和脉冲电子顺磁共振方法，我们确定了放置在二聚体动力蛋白每个颈连接体中间的两个自旋标签之间距离的分布。在没有微管的情况下，距离以3.3 nm为中心，但分布较宽，宽度为2.7 nm。这种广泛的分布意味着连接体是随机线圈，并且远远超出了晶体结构预期的2.5 nm距离。在存在微管的情况下，发现了两种连接子群体：一种与没有微管的情况相似（以3.3 nm为中心的宽分布），另一种分布较窄，以1.3-2.5 nm为中心。在没有核苷酸但有微管存在的情况下，约40%的连接体位于1.9 nm的距离中心，宽度为1.2 nm；剩余的部分和之前一样在3.3 nm处。这表明颈连接器表现出覆盖1.0到5.0 nm宽距离范围的动态特性。当ATP类似物5′-（β，γ-亚胺）三磷酸腺苷和5′-（γ-硫）三磷酸腺苷存在时，40-50%的自旋呈以1.6 nm为中心的极窄分布，宽度为0.4-0.5 nm。其余种群呈现3.3 nm的宽分布。在这些条件下，大部分连接体牢固地停靠在电机核心或微管上，而其余的则是无序的。我们提出，连接体中核苷酸依赖性的大灵活性变化有助于驱动蛋白分子沿着微管步进8纳米的方向偏差。
Using dipolar continuous-wave and pulsed electron paramagnetic resonance methods, we have determined the distribution of the distances between two spin labels placed on the middle of each of the neck linkers of dimeric kinesin. In the absence of microtubules, the distance was centered at 3.3 nm, but displayed a broad distribution with a width of 2.7 nm. This broad distribution implies that the linkers are random coils and extend well beyond the 2.5-nm distance expected of crystal structures. In the presence of microtubules, two linker populations were found: one similar to that observed in the absence of microtubules (a broad distribution centered at 3.3 nm), and the second population with a narrower distribution centered at 1.3–2.5 nm. In the absence of nucleotide but in the presence of microtubules, ∼40% of the linkers were at a distance centered at 1.9 nm with a 1.2-nm width; the remaining fraction was at 3.3 nm, as before. This suggests that neck linkers exhibit dynamics covering a wide distance range between 1.0 and 5.0 nm. In the presence of ATP analogs adenosine 5′-(β,γ-imido)triphosphate and adenosine 5′-(γ-thio)triphosphate, 40–50% of the spins showed a very narrow distribution centered at 1.6 nm, with a width of 0.4–0.5 nm. The remaining population displayed the broad 3.3-nm distribution. Under these conditions, a large fraction of linkers are docked firmly onto a motor core or microtubule, while the remainder is disordered. We propose that large nucleotide-dependent flexibility changes in the linkers contribute to the directional bias of the kinesin molecule stepping 8 nm along the microtubule.