Modification of Loop 1 Affects the Nucleotide Binding Properties of Myo1c, the Adaptation Motor in the Inner Ear
Modification of Loop 1 Affects the Nucleotide Binding Properties of Myo1c, the Adaptation Motor in the Inner Ear
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DOI：
10.1021/bi901803j
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发表时间：
2010-02-09
期刊：
影响因子：
2.9
通讯作者：
Coluccio, Lynne M.
中科院分区：
文献类型：
--
作者：
Adamek, Nancy;Lieto-Trivedi, Alena;Coluccio, Lynne M.
Myo1c 是肌动蛋白相关分子马达的哺乳动物肌球蛋白 I 家族的八个成员之一。在内车毛细胞的静纤毛中，Myo1c 可能充当适应马达，调节转导通道的打开和关闭。尽管所有肌球蛋白的 N 端运动结构域（包含核苷酸和肌动蛋白结合位点）的序列和结构都具有保守性，但一些差异包括表面环的长度和组成，包括位于核苷酸结合结构域附近的环 1。为了研究环 1 的作用，我们在昆虫细胞中表达了 Myo1c 截短形式的突变体、Myo1c(IIQ)，以及 Myo1c(IIQ) 与其他肌球蛋白的类似环的嵌合体。我们发现，用丙氨酸替换环 1 中的带电残基或用一系列丙氨酸替换整个环不会改变 Myo1c(IIQ) 的 ATP 酶活性、瞬时动力学特性或 Ca2+ 敏感性。将环 1 替换为强直平滑肌肌球蛋白 II (Myo1c(IIQ)-tonic) 的相应区域或替换为单一甘氨酸 (Myo1c(IIQ)-G)，可加速 ADP 在 Ca2+ 中的释放 2-3 倍，而替换为相位肌球蛋白 II (Myo1c(IIQ)-phasic) 的环 1 则可加速 ADP 的释放 35 倍。对含有单个 α 螺旋或 SAH 结构域的嵌合体进行的运动测定表明，Myo1c(SAH)-tonic 在体外易位肌动蛋白的速度是 Myo1c(SAH)-WT 的两倍，是 Myo1c(SAH)-G 的 3 倍。研究表明，通过修饰环 1 在 Myo1c 中诱导的变化与环供体肌球蛋白的行为或之前在类似的 Myo1b 嵌合体中观察到的变化没有相似之处。
Myo1c is one of eight members of the mammalian myosin I family of actin-associated molecular motors. In stereocilia of the hair cells in the inner car, Myo1c presumably serves as the adaptation motor, which regulates the opening and closing of transduction channels. Although there is conservation of sequence and structure among all myosins in the N-terminal motor domain, which contains the nucleotide- and actin-binding sites, some differences include the length and composition of surface loops, including loop 1, which lies near the nucleotide-binding domain. To investigate the role of loop 1, We expressed in insect cells mutants of a truncated form of Myo1c, Myo1c(IIQ), as well as chimeras of Myo1c(IIQ) with the analogous loop from other myosins. We found that replacement of the charged residues in loop 1 with alanines or the whole loop with a series of alanines did not alter the ATPase activity, transient kinetics properties, or Ca2+ sensitivity of Myo1c(IIQ). Substitution of loop 1 with that of the corresponding region from tonic smooth muscle myosin II (Myo1c(IIQ)-tonic) or replacement with a single glycine (Myo1c(IIQ)-G) accelerated the release of ADP from A.M 2-3-fold in Ca2+, whereas substitution with loop 1 from phasic muscle myosin II (Myo1c(IIQ)-phasic) accelerated the release of ADP 35-fold. Motility assays with chimeras containing a single alpha-helix, or SAH, domain showed that Myo1c(SAH)-tonic translocated actin in vitro twice as fast as Myo1c(SAH)-WT and 3-fold faster than Myo1c(SAH)-G. The studies show that changes induced in Myo1c via modification of loop 1 showed no resemblance to the behavior of the loop donor myosins or to the changes previously observed with similar Myo1b chimeras.