The 2.1-Å Crystal Structure of Native Neuroserpin Reveals Unique Structural Elements That Contribute to Conformational Instability
The 2.1-Å Crystal Structure of Native Neuroserpin Reveals Unique Structural Elements That Contribute to Conformational Instability
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DOI：
10.1016/j.jmb.2009.03.007
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发表时间：
2009-04-24
影响因子：
5.6
通讯作者：
Lomas, David A.
中科院分区：
文献类型：
--
作者：
Takehara, Sayaka;Onda, Maki;Lomas, David A.
神经丝氨酸蛋白酶抑制剂是组织型纤溶酶原激活剂（tPA）的选择性抑制剂，其在神经元可塑性、记忆和学习中起重要作用。我们在这里报告的晶体结构的本地人类neuroserpin在2.1埃的分辨率。该结构在股线1B和2B之间具有螺旋反应中心环和Ω环。omega环有助于抑制tPA，因为删除该基序使与tPA的缔合速率常数降低三倍，但对与尿激酶相互作用的动力学没有影响。神经丝氨酸蛋白酶抑制剂的点突变导致有序的细胞内聚合物的形成，这些聚合物是神经丝氨酸蛋白酶抑制剂包涵体痴呆家族性脑病（FENIB）的基础。野生型神经丝氨酸蛋白酶抑制剂也是不稳定的，并且在体外接近生理条件下容易形成聚合物。这部分是由于在位置340处保守的丙氨酸被丝氨酸取代。与野生型neuroserpin相比，用Ala替换Ser 340使熔融温度增加3 ℃，并减少聚合。类似地，神经丝氨酸蛋白酶抑制剂在螺旋F末端具有Asn-Leu-瓦尔，因此与丝氨酸蛋白酶抑制剂的Gly-X-Ile共有序列显著不同。这些氨基酸的共识序列的恢复增加了热稳定性，减少了neuroserpin的聚合和其过渡到潜在的构象。此外，将共有序列引入引起FENIB的S49 P神经丝氨酸蛋白酶抑制剂中增加了突变体的稳定性和抑制活性，以及阻断聚合并增加了重折叠过程中蛋白质的产率。这些数据为神经丝氨酸蛋白酶抑制剂的固有不稳定性和FENIB痴呆的点突变效应提供了分子解释。(C)2009爱思唯尔有限公司保留所有权利。
Neuroserpin is a selective inhibitor of tissue-type plasminogen activator (tPA) that plays an important role in neuronal plasticity, memory and learning. We report here the crystal structure of native human neuroserpin at 2.1 angstrom resolution. The structure has a helical reactive center loop and an omega loop between strands 1B and 2B. The omega loop contributes to the inhibition of tPA, as deletion of this motif reduced the association rate constant with tPA by threefold but had no effect on the kinetics of interaction with urokinase. Point mutations in neuroserpin cause the formation of ordered intracellular polymers that underlie dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type neuroserpin is also unstable and readily forms polymers under near-physiological conditions in vitro. This is, in part, due to the substitution of a conserved alanine for serine at position 340. The replacement of Ser340 by Ala increased the melting temperature by 3 degrees C and reduced polymerization as compared to wild-type neuroserpin. Similarly, neuroserpin has Asn-Leu-Val at the end of helix F and thus differs markedly from the Gly-X-Ile consensus sequence of the serpins. Restoration of these amino acids to the consensus sequence increased thermal stability and reduced the polymerization of neuroserpin and its transition to the latent conformer. Moreover, introduction of the consensus sequence into S49P neuroserpin that causes FENIB increased the stability and inhibitory activity of the mutant, as well as blocked polymerization and increased the yield of protein during refolding. These data provide a molecular explanation for the inherent instability of neuroserpin and the effect of point mutations that underlie the dementia FENIB. (C) 2009 Elsevier Ltd. All rights reserved.