The 2.1-Å Crystal Structure of Native Neuroserpin Reveals Unique Structural Elements That Contribute to Conformational Instability

被引:35
作者
Takehara, Sayaka [2 ]
Onda, Maki [1 ]
Zhang, Juan [1 ]
Nishiyama, Mika [1 ]
Yang, Xiaoyan [1 ]
Mikami, Bunzo [2 ]
Lomas, David A. [3 ]
机构
[1] Osaka Prefecture Univ, Grad Sch Sci, Dept Biol Sci, Naka Ku, Sakai, Osaka 5998570, Japan
[2] Kyoto Univ, Grad Sch Agr, Div Appl Life Sci, Uji 6110011, Japan
[3] Univ Cambridge, Cambridge Inst Med Res, Wellcome Trust MRC, Dept Med, Cambridge CB2 0XY, England
基金
英国医学研究理事会;
关键词
serpin; polymerization; dementia; tPA; metastability; INHIBITORY REACTIVE LOOP; PLASMINOGEN-ACTIVATOR; FORMS POLYMERS; IN-VITRO; ALPHA(1)-ANTITRYPSIN REVEALS; FAMILIAL ENCEPHALOPATHY; PROTEINASE-INHIBITOR; ANGSTROM STRUCTURE; INCLUSION-BODIES; PROTECTS NEURONS;
D O I
10.1016/j.jmb.2009.03.007
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:11 / 20
页数:10
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