Guzik-Lendrum Stephanie, Rank Katherine C, Bensel Brandon M, Taylor Keenan C, Rayment Ivan, Gilbert Susan P
Department of Biological Sciences and Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York.
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin.
Biophys J. 2015 Oct 6;109(7):1472-82. doi: 10.1016/j.bpj.2015.08.004.
Mammalian KIF3AC is classified as a heterotrimeric kinesin-2 that is best known for organelle transport in neurons, yet in vitro studies to characterize its single molecule behavior are lacking. The results presented show that a KIF3AC motor that includes the native helix α7 sequence for coiled-coil formation is highly processive with run lengths of ∼1.23 μm and matching those exhibited by conventional kinesin-1. This result was unexpected because KIF3AC exhibits the canonical kinesin-2 neck-linker sequence that has been reported to be responsible for shorter run lengths observed for another heterotrimeric kinesin-2, KIF3AB. However, KIF3AB with its native neck linker and helix α7 is also highly processive with run lengths of ∼1.62 μm and exceeding those of KIF3AC and kinesin-1. Loop L11, a component of the microtubule-motor interface and implicated in activating ADP release upon microtubule collision, is significantly extended in KIF3C as compared with other kinesins. A KIF3AC encoding a truncation in KIF3C loop L11 (KIF3ACΔL11) exhibited longer run lengths at ∼1.55 μm than wild-type KIF3AC and were more similar to KIF3AB run lengths, suggesting that L11 also contributes to tuning motor processivity. The steady-state ATPase results show that shortening L11 does not alter kcat, consistent with the observation that single molecule velocities are not affected by this truncation. However, shortening loop L11 of KIF3C significantly increases the microtubule affinity of KIF3ACΔL11, revealing another structural and mechanistic property that can modulate processivity. The results presented provide new, to our knowledge, insights to understand structure-function relationships governing processivity and a better understanding of the potential of KIF3AC for long-distance transport in neurons.
哺乳动物的KIF3AC被归类为异源三聚体驱动蛋白-2，它在神经元的细胞器运输方面最为人所知，但目前缺乏对其单分子行为进行表征的体外研究。本文给出的结果表明，包含用于形成卷曲螺旋的天然α7螺旋序列的KIF3AC马达具有高度的持续性，运行长度约为1.23μm，与传统驱动蛋白-1的表现相当。这一结果出乎意料，因为KIF3AC具有典型的驱动蛋白-2颈链序列，据报道该序列导致另一种异源三聚体驱动蛋白-2 KIF3AB的运行长度较短。然而，具有天然颈链和α7螺旋的KIF3AB也具有高度的持续性，运行长度约为1.62μm，超过了KIF3AC和驱动蛋白-1。L11环是微管-马达界面的一个组成部分，与微管碰撞时激活ADP释放有关，与其他驱动蛋白相比，在KIF3C中显著延长。编码KIF3C环L11截短的KIF3AC（KIF3ACΔL11）的运行长度约为1.55μm，比野生型KIF3AC更长，且更接近KIF3AB的运行长度，这表明L11也有助于调节马达的持续性。稳态ATP酶结果表明，缩短L11不会改变催化常数，这与单分子速度不受该截短影响的观察结果一致。然而，缩短KIF3C的L11环显著增加了KIF3ACΔL11对微管的亲和力，揭示了另一种可调节持续性的结构和机制特性。据我们所知，本文给出的结果为理解控制持续性的结构-功能关系提供了新的见解，并有助于更好地理解KIF3AC在神经元中进行长距离运输的潜力。
