Discovery of an allosteric site in the caspases
Discovery of an allosteric site in the caspases
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DOI：
10.1073/pnas.0404781101
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发表时间：
2004-08-24
影响因子：
11.1
通讯作者：
Wells, JA
中科院分区：
文献类型：
--
作者：
Hardy, JA;Lam, J;Wells, JA
通过构象变化对蛋白质进行变构调节是生物控制的主要手段。传统上，它一直很难确定和表征新的变构位点和配体，冻结这些构象状态。我们提出了一个定点的方法，利用拴系捕获抑制性小分子在网站远离活性位点的可逆二硫键形成。我们针对蛋白酶的半胱天冬酶家族的两个成员（半胱天冬酶-3和半胱天冬酶-7）的可接近半胱氨酸筛选了10，000种含巯基化合物的文库。我们发现了一个以前未报道的和保守的变构位点在一个深腔中的二聚体界面14 A的活性位点。该位点含有天然半胱氨酸，当与两种特定化合物中的任何一种形成二硫键时，会使这些蛋白酶失活。变构位点与活性位点功能性偶联，使得化合物在变构位点的结合防止肽在活性位点的结合。由任一化合物结合的半胱天冬酶-7的X射线晶体结构表明，它们通过捕获酶原样构象来抑制半胱天冬酶-7。这种方法可能是有用的，以确定新的变构位点从天然或工程半胱氨酸，研究蛋白质的变构转换，并核药物发现的努力。
Allosteric regulation of proteins by conformational change is a primary means of biological control. Traditionally it has been difficult to identify and characterize novel allosteric sites and ligands that freeze these conformational states. We present a site-directed approach using Tethering for trapping inhibitory small molecules at sites away from the active site by reversible disulfide bond formation. We screened a library of 10,000 thiol-containing compounds against accessible cysteines of two members of the caspase family of proteases, caspase-3 and -7. We discovered a previously unreported and conserved allosteric site in a deep cavity at the dimer interface 14 A from the active site. This site contains a natural cysteine that, when disulfide-bonded with either of two specific compounds, inactivates these proteases. The allosteric site is functionally coupled to the active site, such that binding of the compounds at the allosteric site prevents peptide binding at the active site. The x-ray crystal structures of caspase-7 bound by either compound demonstrates that they inhibit caspase-7 by trapping a zymogen-like conformation. This approach may be useful to identify new allosteric sites from natural or engineered cysteines, to study allosteric transitions in proteins, and to nucleate drug discovery efforts.