A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility.
A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility.
复制标题
环肽丝氨酸蛋白酶抑制剂：通过增加肽灵活性来增加亲和力
DOI：
10.1371/journal.pone.0115872
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Andreasen PA
中科院分区：
文献类型：
--
作者：
Zhao B;Xu P;Jiang L;Paaske B;Kromann-Hansen T;Jensen JK;Sørensen HP;Liu Z;Nielsen JT;Christensen A;Hosseini M;Sørensen KK;Nielsen NC;Jensen KJ;Huang M;Andreasen PA
肽作为蛋白酶抑制剂正引起越来越多的兴趣。在这里，我们展示了一种新的抑制机制和一种新的外源相互作用，用于噬菌体展示的肽库衍生的丝氨酸蛋白酶小鼠尿激酶型纤溶酶原激活物（uPA）的竞争性抑制剂，mupain1 （CPAYSRYLDC）。我们使用了x射线晶体结构分析、定点诱变、液态核磁共振、表面等离子体共振分析、等温滴定量热法和野生型和工程变异的小鼠和人类uPA。我们证明Arg6插入到S1特异性口袋中，其羰基相对于Ser195和氧阴离子空洞排列不正确，解释了为什么肽是抑制剂而不是底物。用具有脂肪或芳香环结构的新型非天然精氨酸类似物取代P1精氨酸导致亲和性增加，这取决于P1 - S1和外源相互作用的变化。位点定向诱变表明，外源相互作用虽然仍然支持高亲和力结合，但在不同的uPA变体之间存在很大差异。令人惊讶的是，高亲和结合被ala取代的Asp9的肽促进，尽管不太有利的结合熵和极性相互作用的损失。我们得出的结论是，多肽灵活性的增加允许更有利的外源相互作用，结合使用新的Arg类似物作为P1残基，可以用来操纵这种肽抑制剂的亲和力和特异性，这与通过减少熵负担来提高抑制剂亲和力的传统尝试不同。
Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
14.8
作者：
Heinis, Christian;Rutherford, Trevor;Winter, Greg
通讯作者：
Winter, Greg
影响因子：
3.6
作者：
Hosseini, Masood;Jiang, Longguang;Jensen, Knud J.
通讯作者：
Jensen, Knud J.
影响因子：
4
作者：
Angelini, Alessandro;Cendron, Laura;Heinis, Christian
通讯作者：
Heinis, Christian
影响因子：
120.1
作者：
Drag, Marcin;Salvesen, Guy S.
通讯作者：
Salvesen, Guy S.