Identification of a minimal myosin Va binding site within an intrinsically unstructured domain of melanophilin
Identification of a minimal myosin Va binding site within an intrinsically unstructured domain of melanophilin
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DOI：
10.1074/jbc.m701932200
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发表时间：
2007-07-20
影响因子：
4.8
通讯作者：
Spudich, James A.
中科院分区：
文献类型：
--
作者：
Geething, Nathan C.;Spudich, James A.
肌球蛋白V是一种分子马达，可以运输各种细胞货物，包括细胞器，囊泡和信使RNA。黑素体是一种致密的含有色素的细胞器，其周围的适当分布依赖于肌动蛋白和肌球蛋白Va的活性。肌球蛋白Va向黑素体的募集和黑素体的适当转运需要亲黑素蛋白，其直接结合肌球蛋白Va并通过Rab 27 a栓系到黑素体膜。在这里，我们使用高度纯化的蛋白质来证明，球状尾域的肌球蛋白Va直接结合到一个本质上非结构化的结构域的黑素。亲黑素的肌球蛋白Va结合域缺乏稳定的二级结构，并且H-1 NMR测量表明该蛋白质是未折叠的。该结构域对温和的蛋白水解极其敏感，并且具有与随机卷曲样多肽一致的流体动力学半径。我们发现，肌球蛋白Va结合不诱导全球折叠的黑素。利用截短的亲黑素来定义亲黑素内的短肽序列（26个残基），其对于肌球蛋白Va结合是关键的。我们证明，对应于这些残基的肽直接结合到球状尾结构域具有相同的亲黑素的亲和力。我们讨论了蛋白质内在紊乱在黑素体膜上肌球蛋白Va的募集和维持中的可能意义。
Myosin V is a molecular motor that transports a variety of cellular cargo, including organelles, vesicles, and messenger RNA. The proper peripheral distribution of melanosomes, a dense pigment-containing organelle, is dependent on actin and the activity of myosin Va. The recruitment of myosin Va to the melanosome and proper transport of the melanosome requires melanophilin, which directly binds to myosin Va and is tethered to the melanosome membrane via Rab27a. Here we use highly purified proteins to demonstrate that the globular tail domain of myosin Va binds directly to an intrinsically unstructured domain of melanophilin. The myosin Va binding domain of melanophilin lacks stable secondary structure, and H-1 NMR measurements indicate that the protein is unfolded. This domain is extremely sensitive to mild proteolysis and has a hydrodynamic radius that is consistent with a random coil-like polypeptide. We show that myosin Va binding does not induce the global folding of melanophilin. Truncations of melanophilin were utilized to define a short peptide sequence (26 residues) within melanophilin that is critical for myosin Va binding. We demonstrate that a peptide corresponding to these residues binds directly to the globular tail domain with the same affinity as melanophilin. Wediscuss the possible implications of protein intrinsic disorder in recruitment and maintenance of myosin Va on melanosome membranes.