Uncoupling Kapβ2 substrate dissociation and ran binding
Uncoupling Kapβ2 substrate dissociation and ran binding
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DOI：
10.1021/bi012122p
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发表时间：
2002-06-04
期刊：
影响因子：
2.9
通讯作者：
Blobel, G
中科院分区：
文献类型：
--
作者：
Chook, YM;Jung, A;Blobel, G
Karyopherinbeta2（Kap beta2）将多种mRNA结合蛋白输入细胞核。输入底物在细胞核中的释放涉及高亲和力Kapbeta2-RanGTP复合物的形成和输入底物的伴随解离。Kapbeta2-RanGppNHp复合物的晶体结构表明，Ran结合在Kapbeta2的N-末端弓和底物最有可能结合其C-末端arch.The结构提出了一种机制RAN介导的底物解离，其中一个长的内部酸性环在Kapbeta2中传输的GTP酶和底物位点之间的结构信息，导致底物的位移环时，Ran被绑定。为了研究底物解离的分子机制，我们蛋白水解地切割了Kapbeta2的酸性环（cl-Kapbeta2），并且构建了具有截短环的Kapbeta2的突变体（TL-Kapbeta2）。两种修饰的Kapbeta2都不能进行Ran介导的底物解离。我们还绘制了底物mRNA结合蛋白A1的Kapbeta2结合位点的边界，使用广泛适用的方法，采用NMR光谱。这允许设计试剂以定量Kap β 2蛋白对Ran和底物的亲和力。cl-Kapbeta2、TL-Kapbeta2和天然Kapbeta2对RanGppNHp和输入底物具有相当的亲和力，表明环的扰动没有改变二元Kapbeta2-Ran或Kapbeta2-底物相互作用的强度。TL-Kapbeta2突变体还同时结合RanGppNHP和底物以形成三元复合物，这表明除了Ran结合和底物解离之间的偶联丧失之外，Kapbeta2上的两个配体位点在空间上是不同的。TL-Kapbeta2突变体中Ran结合和底物解离的解偶联在毛地黄苷透化的HeLa细胞中Ran介导的荧光底物核摄取的显着丧失中进一步明显。这些结果支持我们先前提出的GTP酶介导的Kapbeta2-底物解离机制，其中Kapbeta2的酸性环物理耦合不同的Ran和底物结合位点。
Karyopherinbeta2 (Kapbeta2) imports a variety of mRNA binding proteins into the nucleus. Release of import substrates in the nucleus involves formation of a high-affinity Kapbeta2-RanGTP complex and concomitant dissociation of import substrates. The crystal structure of the Kapbeta2-RanGppNHp complex shows that Ran binds in the Kapbeta2 N-terminal arch and substrate most likely binds its C-terminal arch. The structure suggested a mechanism for Ran-mediated substrate dissociation where a long internal acidic loop in Kapbeta2 transmits structural information between the GTPase and substrate sites, leading to displacement of substrate by the loop when Ran is bound. To study the molecular mechanism of substrate dissociation, we have cleaved the acidic loop of Kapbeta2 proteolytically (cl-Kapbeta2) and also constructed a mutant of Kapbeta2 with a truncated loop (TL-Kapbeta2). Both modified Kapbeta2s are unable to undergo Ran-mediated substrate dissociation. We have also mapped the boundaries of the Kapbeta2 binding site of substrate mRNA binding protein A1 using a widely applicable method employing NMR spectroscopy. This has allowed design of reagents to quantitate the affinities of the Kapbeta2 proteins for Ran and substrate. cl-Kapbeta2, TL-Kapbeta2, and native Kapbeta2 have comparable affinities for both RanGppNHp and import substrates, indicating that perturbation of the loop has not altered the strength of binary Kapbeta2-Ran or Kapbeta2-substrate interactions. The TL-Kapbeta2 mutant also binds RanGppNHP and substrate simultaneously to form a ternary complex, indicating that in addition to the loss of coupling between Ran binding and substrate dissociation, the two ligand sites on Kapbeta2 are spatially distinct. The uncoupling of Ran binding and substrate dissociation in the TL-Kapbeta2 mutant is further evident in significant loss of Ran-mediated nuclear uptake of fluorescent substrate in digitonin-permeabilized HeLa cells. These results support our previously proposed GTPase-mediated Kapbeta2-substrate dissociation mechanism where the acidic loop of Kapbeta2 physically couples distinct Ran and substrate binding sites.