Dissecting an Allosteric Switch in Caspase-7 Using Chemical and Mutational Probes
Dissecting an Allosteric Switch in Caspase-7 Using Chemical and Mutational Probes
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DOI：
10.1074/jbc.m109.001826
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发表时间：
2009-09-18
影响因子：
4.8
通讯作者：
Wells, James A.
中科院分区：
文献类型：
--
作者：
Hardy, Jeanne A.;Wells, James A.
凋亡 caspase，例如 caspase-7，被储存为无活性的蛋白酶酶原，当被激活时，会导致命运决定开关以诱导细胞死亡。我们之前发现了含硫醇的小分子抑制剂，当它们被束缚时，会显示出变构位点并捕获与酶的酶原形式相似的构象。我们注意到这些化合物诱导的三个结构转变：（i）变构位点中 Tyr-223 和 Arg-187 之间相互作用的破坏，这阻止了催化半胱氨酸的正确排序； (ii) 将 L2' 环钉在变构位点上，这阻止了底物结合槽正确排序的关键相互作用； (iii) 位于催化 Cys-186 和 Arg-187 之后的 Gly-188 处的铰链式旋转。在这里，我们报告了这些区域的系统突变分析，以剖析它们在介导这些化合物诱导的变构转变方面的功能重要性。将铰链 Gly-188 突变为限制性脯氨酸会导致催化效率大幅降低 6000 倍。 Arg-187-Tyr-223 对中的突变的影响要小得多（减少 3-20 倍）。有趣的是，尽管变构对突变体仍然允许结合和变构抑制，但它们部分缓解了活性位点和变构位点抑制剂之间结合的相互排他性。这些数据突出显示了一小部分残基，这些残基对于介导从活性酶原状态向非活性酶原状态的转变至关重要。
Apoptotic caspases, such as caspase-7, are stored as inactive protease zymogens, and when activated, lead to a fate-determining switch to induce cell death. We previously discovered small molecule thiol-containing inhibitors that when tethered revealed an allosteric site and trapped a conformation similar to the zymogen form of the enzyme. We noted three structural transitions that the compounds induced: (i) breaking of an interaction between Tyr-223 and Arg-187 in the allosteric site, which prevents proper ordering of the catalytic cysteine; (ii) pinning the L2' loop over the allosteric site, which blocks critical interactions for proper ordering of the substrate-binding groove; and (iii) a hinge-like rotation at Gly-188 positioned after the catalytic Cys-186 and Arg-187. Here we report a systematic mutational analysis of these regions to dissect their functional importance to mediate the allosteric transition induced by these compounds. Mutating the hinge Gly-188 to the restrictive proline causes a massive similar to 6000-fold reduction in catalytic efficiency. Mutations in the Arg-187-Tyr-223 couple have a far less dramatic effect (3-20-fold reductions). Interestingly, although the allosteric couple mutants still allow binding and allosteric inhibition, they partially relieve the mutual exclusivity of binding between inhibitors at the active and allosteric sites. These data highlight a small set of residues critical for mediating the transition from active to inactive zymogen-like states.