Interactions of Plasminogen Activator Inhibitor-1 with Vitronectin Involve an Extensive Binding Surface and Induce Mutual Conformational Rearrangements
Interactions of Plasminogen Activator Inhibitor-1 with Vitronectin Involve an Extensive Binding Surface and Induce Mutual Conformational Rearrangements
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DOI：
10.1021/bi8017015
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发表时间：
2009-03-03
期刊：
影响因子：
2.9
通讯作者：
Andreasen, Peter A.
中科院分区：
文献类型：
--
作者：
Blouse, Grant E.;Dupont, Daniel M.;Andreasen, Peter A.
为了探索纤溶酶原激活物抑制剂-1（派-1）与其辅因子玻连蛋白的关联过程中的早期事件，我们采用了一种强大的策略，结合蛋白质工程，荧光光谱和快速反应动力学。设计用于监测PAT-1与玻连蛋白的快速结合的荧光停流实验表明派-1与天然玻连蛋白的快速、浓度依赖性、双相结合，但仅与生长调节蛋白B（SMB）结构域的非相结合，表明仅当存在全长玻连蛋白时才发生多相结合相互作用。尽管如此，在所有情况下，最初的快速相互作用之后是较慢的荧光变化归因于派-1的构象变化。使用具有非天然氨基酸的工程化的荧光沉默派-1的补充实验表明，伴随的结构变化也发生在天然玻连蛋白中。此外，我们已经测量了玻连蛋白在与靶蛋白酶反应期间对PAT-1的反应中心环插入折叠A的速率的影响。与各种派-1的变体，我们观察到，全长玻连蛋白和SMB域有蛋白酶特异性的环插入率的影响，但这两个表现出明显不同的效果。这些结果支持派-1与玻连蛋白结合的模型，其中相互作用表面延伸超出由SMB结构域占据的派-1区域。支持该模型的是最近的结果，其定义了位于生长调节蛋白B结构域之外的玻连蛋白上的派-1结合位点（Schar，C. R.，布卢斯湾E、小K H、和Peterson，C. B。（2008）J.BioLChem.283，10297-10309）和派-1上的互补位点（Schar，C. R.，詹森，J. K.，克里斯滕森，A.，布卢斯湾E、Andreasen，P. A.，和Peterson，C. B。（2008）J.Biol.Chem.283，28487-28496）。
In order to explore early events during the association of plasminogen activator inhibitor-1 (PAI-1) with its cofactor vitronectin, we have applied a robust strategy that combines protein engineering, fluorescence spectroscopy, and rapid reaction kinetics. Fluorescence stopped-flow experiments designed to monitor the rapid association of PAT-1 with vitronectin indicate a fast, concentration-dependent, biphasic binding of PAI-1 to native vitronectin but only a rnonophasic association with the somatomedin B (SMB) domain, suggesting that multiple phases of the binding interaction occur only when full-length vitronectin is present. Nonetheless, in all cases, the initial fast interaction is followed by slower fluorescence changes attributed to a conformational change in PAI-1. Complementary experiments using an engineered, fluorescendy silent PAI-1 with non-natural amino acids showed that concomitant structural changes occur as well in native vitronectin. Furthermore, we have measured the effect of vitronectin on the rate of insertion of the reactive center loop into-sheet A of PAT-1 during reaction with target proteases. With a variety of PAI-1 variants, we observe that both full-length vitronectin and the SMB domain have protease-specific effects on the rate of loop insertion but that the two exhibit clearly different effects. These results support a model for PAI-1 binding to vitronectin in which the interaction surface extends beyond the region of PAI-1 occupied by the SMB domain. In support of this model are recent results that define a PAI-1-binding site on vitronectin that lies outside the somatomedin B domain (Schar, C. R., Blouse, G. E., Minor, K. H., and Peterson, C. B. (2008) J. BioL Chem. 283, 10297-10309) and the complementary site on PAI-1 (Schar, C. R., Jensen, J. K., Christensen, A., Blouse, G. E., Andreasen, P. A., and Peterson, C. B. (2008) J. Biol. Chem. 283, 28487-28496).