The Binding Mechanism of a Peptidic Cyclic Serine Protease Inhibitor
The Binding Mechanism of a Peptidic Cyclic Serine Protease Inhibitor
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肽类环状丝氨酸蛋白酶抑制剂的结合机制
DOI：
10.1016/j.jmb.2011.07.028
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发表时间：
2011-09-16
影响因子：
5.6
通讯作者：
Andreasen, Peter A.
中科院分区：
文献类型：
--
作者：
Jiang, Longguang;Svane, Anna S. P.;Andreasen, Peter A.
丝氨酸蛋白酶是研究催化和抑制机制的经典对象，也是令人感兴趣的治疗靶点。由于小分子丝氨酸蛋白酶抑制剂通常存在特异性问题，从噬菌体展示肽库中分离的肽抑制剂引起了人们的极大关注。在这里，我们研究了肽抑制剂结合丝氨酸蛋白酶靶点的机制。我们的模型是upain-1（CSWRGLENHRMC），一种二硫键约束的竞争性抑制剂的人尿激酶型纤溶酶原激活剂的非经典抑制机制和异常高的特异性。使用一些修改后的变体的upain-1，我们的特点是upain-1-尿激酶型纤溶酶原激活剂复合物，使用X-射线晶体结构分析，确定了一个模型的肽在溶液中的NMR光谱，并分析结合动力学和热力学的表面等离子体共振和等温滴定量热法。我们发现，upain-1改变了主链构象和侧链的方向，因为它结合到蛋白酶，特别是它的Trp 3残基和周围的骨干。upain-1的性质受到向核心、二硫键约束序列添加三到四个氨基酸长的N-末端和C-末端延伸的强烈影响：与单独的核心肽相比，C-末端延伸稳定溶液结构，并且肽的蛋白酶结合结构通过N-末端延伸和Trp 3周围的核心肽之间的肽内接触稳定。这些结果提供了一个独特的详细描述的肽蛋白酶抑制剂的结合到其目标，并在开发具有高特异性和新的抑制机制的肽抑制剂具有普遍的重要性。(C)2011爱思唯尔有限公司保留所有权利。
Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding kinetics and thermodynamics by surface plasmon resonance and isothermal titration calorimetry. We found that upain-1 changes both main-chain conformation and side-chain orientations as it binds to the protease, in particular its Trp3 residue and the surrounding backbone. The properties of upain-1 are strongly influenced by the addition of three to four amino acids long N-terminal and C-terminal extensions to the core, disulfide-bond-constrained sequence: The C-terminal extension stabilises the solution structure compared to the core peptide alone, and the protease-bound structure of the peptide is stabilised by intrapeptide contacts between the N-terminal extension and the core peptide around Trp3. These results provide a uniquely detailed description of the binding of a peptidic protease inhibitor to its target and are of general importance in the development of peptidic inhibitors with high specificity and new inhibitory mechanisms. (C) 2011 Elsevier Ltd. All rights reserved.