Calcium-bound structure of calpain and its mechanism of inhibition by calpastatin
Calcium-bound structure of calpain and its mechanism of inhibition by calpastatin
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DOI：
10.1038/nature07451
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发表时间：
2008-11-20
期刊：
影响因子：
64.8
通讯作者：
Davies, Peter L.
中科院分区：
文献类型：
--
作者：
Hanna, Rachel A.;Campbell, Robert L.;Davies, Peter L.
钙蛋白酶是一种非溶酶体钙依赖的半胱氨酸蛋白酶，它响应钙信号选择性地裂解蛋白质(1)，从而控制细胞功能，如细胞骨架重塑、细胞周期进程、基因表达和细胞凋亡(2-4)。在哺乳动物中，钙蛋白酶家族中两个特征最好的成员，即钙蛋白1和钙蛋白2(分别为u-钙蛋白和m-钙蛋白)在哺乳动物中普遍表达。钙蛋白酶的活性受到内源性抑制物钙调蛋白的严格控制，这是一种内在的非结构蛋白，能够可逆地结合和抑制四个钙蛋白酶分子，但只有在存在钙的情况下(5，6)。到目前为止，Calastatin的抑制机制和其绝对特异性的基础仍然是推测的(7-9)。目前还不清楚这种非结构蛋白是如何在不被自身切割的情况下抑制钙调蛋白的，也不知道钙是如何诱导促进钙调蛋白与钙调蛋白结合的变化的。在这里，我们报道了钙结合的钙蛋白酶2异二聚体的2.4埃分辨率的晶体结构，该二聚体被钙蛋白酶的四个抑制结构域之一结合。钙调蛋白被认为是通过占据活性部位裂隙的两侧来抑制钙蛋白酶的。虽然抑制物穿过活性部位的裂解，但它以一种新颖的方式绕过活性部位半胱氨酸而逃避切割。Calastatin的抑制域识别多个仅以钙结合形式存在的低亲和力位点，导致一种紧密、特异和钙依赖的相互作用。这种晶体结构和相关的络合物(10)的晶体结构也揭示了Calain与钙结合时所经历的构象变化，包括活性部位裂解的打开和结构域相互之间的移动，以产生更紧密的酶。
Calpains are non-lysosomal calcium-dependent cysteine proteinases that selectively cleave proteins in response to calcium signals(1) and thereby control cellular functions such as cytoskeletal remodelling, cell cycle progression, gene expression and apoptotic cell death(2-4). In mammals, the two best-characterized members of the calpain family, calpain 1 and calpain 2 ( mu-calpain and m-calpain, respectively), are ubiquitously expressed. The activity of calpains is tightly controlled by the endogenous inhibitor calpastatin, which is an intrinsically unstructured protein capable of reversibly binding and inhibiting four molecules of calpain, but only in the presence of calcium(5,6). To date, the mechanism of inhibition by calpastatin and the basis for its absolute specificity have remained speculative(7-9). It was not clear how this unstructured protein inhibits calpains without being cleaved itself, nor was it known how calcium induced changes that facilitated the binding of calpastatin to calpain. Here we report the 2.4-angstrom-resolution crystal structure of the calcium-bound calpain 2 heterodimer bound by one of the four inhibitory domains of calpastatin. Calpastatin is seen to inhibit calpain by occupying both sides of the active site cleft. Although the inhibitor passes through the active site cleft it escapes cleavage in a novel manner by looping out and around the active site cysteine. The inhibitory domain of calpastatin recognizes multiple lower affinity sites present only in the calcium-bound form of the enzyme, resulting in an interaction that is tight, specific and calcium dependent. This crystal structure, and that of a related complex(10), also reveal the conformational changes that calpain undergoes on binding calcium, which include opening of the active site cleft and movement of the domains relative to each other to produce a more compact enzyme.