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首页> 外文期刊>Journal of Molecular Biology >The binding mechanism of a peptidic cyclic serine protease inhibitor.
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The binding mechanism of a peptidic cyclic serine protease inhibitor.

机译：肽环丝氨酸蛋白酶抑制剂的结合机制。

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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.
机译：丝氨酸蛋白酶是研究催化和抑制机理以及作为治疗靶标的经典对象。由于小分子丝氨酸蛋白酶抑制剂通常存在特异性问题，因此从噬菌体展示的肽库中分离出的肽类抑制剂引起了极大的关注。在这里，我们已经研究了肽抑制剂与丝氨酸蛋白酶靶标结合的机制。我们的模型是upain-1（CSWRGLENHRMC），它是人尿激酶型纤溶酶原激活剂的二硫键约束竞争性抑制剂，具有非典型的抑制机制和异常高的特异性。使用upain-1的许多修饰变体，我们使用X射线晶体结构分析表征了upain-1-尿激酶型纤溶酶原激活物复合物，通过NMR光谱确定了溶液中肽的模型，并分析了结合动力学和热力学通过表面等离振子共振和等温滴定热分析。我们发现，upain-1与蛋白酶结合时，尤其是其Trp3残基和周围的骨架，都改变了主链构象和侧链方向。 upain-1的性质受核心二硫键约束序列中三到四个氨基酸长的N末端和C末端延伸的强烈影响：与C末端相比，C末端延伸稳定了溶液的结构。 N末端延伸区和Trp3周围的核心肽之间的内肽接触使肽结合蛋白酶的结构稳定。这些结果提供了肽蛋白酶抑制剂与其靶标结合的独特详细描述，并且在开发具有高特异性和新抑制机制的肽抑制剂中具有普遍意义。

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