Identification of protease exosite-interacting peptides that enhance substrate cleavage kinetics
Identification of protease exosite-interacting peptides that enhance substrate cleavage kinetics
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DOI：
10.1515/hsz-2012-0162
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发表时间：
2012-09-01
影响因子：
3.7
通讯作者：
Daugherty, Patrick S.
中科院分区：
文献类型：
--
作者：
Jabaiah, Abeer M.;Getz, Jennifer A.;Daugherty, Patrick S.
许多肽酶被认为需要非活性位点相互作用表面或外切位点，以高特异性和催化效率识别和切割生理底物。然而，蛋白酶外切位点的存在和功能仍然模糊，由于缺乏有效的方法来识别和表征外切位点相互作用的底物。为了满足这一需求，我们修改了肽底物的细胞文库（CLiPS）方法，以能够发现与外来位点相互作用的肽配体。由凝血酶和半胱天冬酶-7的活性位点识别的不变切割基序显示在邻近候选外切位点相互作用肽的细菌外表面上。然后使用双色流式细胞术筛选外切位点肽文库中加速活性位点识别基序切割的配体。外位点CLiPS（eCLiPS）鉴定了凝血酶的外位点结合肽，其与凝血酶底物蛋白酶激活受体1中的关键外位点相互作用基序高度相似。蛋白酶活性探针结合外切位点结合肽裂解速度比没有外切位点配体的底物快10倍，增加了它们在体外对凝血酶活性的敏感性。为了比较，用胱天蛋白酶-7筛选产生适度增强（两倍）底物裂解速率的肽。eCLiPS方法提供了一种新的工具来分析蛋白酶外切位点的配体特异性并开发改进的底物。
Many peptidases are thought to require non-active site interaction surfaces, or exosites, to recognize and cleave physiological substrates with high specificity and catalytic efficiency. However, the existence and function of protease exosites remain obscure owing to a lack of effective methods to identify and characterize exosite-interacting substrates. To address this need, we modified the cellular libraries of peptide substrates (CLiPS) methodology to enable the discovery of exosite-interacting peptide ligands. Invariant cleavage motifs recognized by the active sites of thrombin and caspase-7 were displayed on the outer surface of bacteria adjacent to a candidate exosite-interacting peptide. Exosite peptide libraries were then screened for ligands that accelerate cleavage of the active site recognition motif using two-color flow cytometry. Exosite CLiPS (eCLiPS) identified exosite-binding peptides for thrombin that were highly similar to a critical exosite interaction motif in the thrombin substrate, protease-activated receptor 1. Protease activity probes incorporating exosite-binding peptides were cleaved ten-fold faster than substrates without exosite ligands, increasing their sensitivity to thrombin activity in vitro. For comparison, screening with caspase-7 yielded peptides that modestly enhanced (two-fold) substrate cleavage rates. The eCLiPS method provides a new tool to profile the ligand specificity of protease exosites and to develop improved substrates.