Singh Nimisha, Blobel Günter, Shi Hang
Howard Hughes Medical Institute and Laboratory of Cell Biology, The Rockefeller University, New York, NY 10065.
Howard Hughes Medical Institute and Laboratory of Cell Biology, The Rockefeller University, New York, NY 10065
Proc Natl Acad Sci U S A. 2015 Jan 6;112(1):142-7. doi: 10.1073/pnas.1423194112. Epub 2014 Dec 22.
The segregation of approximately two dozen distinct mRNAs from yeast mother to daughter cell cytoplasm is a classical paradigm for eukaryotic mRNA transport. The information for transport resides in an mRNA element 40-100 nt in length, known as "zipcode." Targeted transport requires properly positioned actin filaments and cooperative loading of mRNA cargo to myosin. Cargo loading to myosin uses myosin 4 protein (Myo4p), swi5p-dependent HO expression 2 protein (She2p) and 3 protein (She3p), and zipcode. We previously determined a crystal structure of Myo4p and She3p, their 1:2 stoichiometry and interactome; we furthermore showed that the motor complex assembly requires two Myo4p⋅She3p heterotrimers, one She2p tetramer, and at least a single zipcode to yield a stable complex of [Myo4p⋅She3p⋅She2p⋅zipcode] in 2:4:4:1 stoichiometry in vitro. Here, we report a structure at 2.8-Å resolution of a cocrystal of a She2p tetramer bound to a segment of She3p. In this crystal structure, the She3p segment forms a striking hook that binds to a shallow hydrophobic pocket on the surface of each She2p subunit of the tetramer. Both She3p hook and cognate She2p binding pocket are composed of highly conserved residues. We also discovered a highly conserved region of She3p upstream of its hook region. Because this region consists of basic and aromatic residues, it likely represents part of She3p's binding activity for zipcode. Because She2p also exhibits zipcode-binding activity, we suggest that "hooking" She3p onto She2p aligns each of their zipcode-binding activities into a high-affinity site, thereby linking motor assembly to zipcode.
将大约二十几种不同的mRNA从酵母母细胞分离到子细胞细胞质中是真核生物mRNA运输的经典范例。运输信息存在于一个长度为40 - 100个核苷酸的mRNA元件中，称为“邮政编码”。靶向运输需要正确定位的肌动蛋白丝以及将mRNA货物协同装载到肌球蛋白上。将货物装载到肌球蛋白上需要肌球蛋白4蛋白（Myo4p）、依赖于swi5p的HO表达2蛋白（She2p）和3蛋白（She3p）以及“邮政编码”。我们之前确定了Myo4p和She3p的晶体结构、它们1:2的化学计量比和相互作用组；此外，我们还表明，马达复合体组装需要两个Myo4p·She3p异源三聚体、一个She2p四聚体和至少一个“邮政编码”，以在体外产生化学计量比为2:4:4:1的稳定的[Myo4p·She3p·She2p·“邮政编码”]复合体。在此，我们报告了与一段She3p结合的She2p四聚体共晶体在2.8 Å分辨率下的结构。在这个晶体结构中，She3p片段形成了一个引人注目的钩子，它与四聚体中每个She2p亚基表面上的一个浅疏水口袋结合。She3p钩子和同源的She2p结合口袋均由高度保守的残基组成。我们还在She3p钩子区域上游发现了一个高度保守的区域。由于该区域由碱性和芳香族残基组成，它可能代表了She3p与“邮政编码”结合活性的一部分。因为She2p也表现出“邮政编码”结合活性，我们认为将She3p“钩”到She2p上会使它们各自的“邮政编码”结合活性排列成一个高亲和力位点，从而将马达组装与“邮政编码”联系起来。
