Solution Structure of the Squash Aspartic Acid Proteinase Inhibitor (SQAPI) and Mutational Analysis of Pepsin Inhibition
Solution Structure of the Squash Aspartic Acid Proteinase Inhibitor (SQAPI) and Mutational Analysis of Pepsin Inhibition
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DOI：
10.1074/jbc.m110.137018
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发表时间：
2010-08-27
影响因子：
4.8
通讯作者：
Pascal, Steven M.
中科院分区：
文献类型：
--
作者：
Headey, Stephen J.;MacAskill, Ursula K.;Pascal, Steven M.
南瓜天冬氨酸蛋白酶抑制剂（SQAPI）是一种来自南瓜的蛋白质类蛋白酶抑制剂，是一系列天冬氨酸蛋白酶的有效抑制剂。蛋白质天冬氨酸蛋白酶抑制剂在自然界中是罕见的。植物中唯一的另一个例子可能是从前体丝氨酸蛋白酶抑制剂进化而来。基于序列同源性建模的早期工作表明SQAPI从祖先胱抑素进化而来。在这项工作中，我们使用NMR确定了SQAPI的溶液结构，并表明SQAPI与植物半胱氨酸蛋白酶抑制剂具有相同的折叠。该结构的特征在于四股反平行β-片层以类似于手握网球拍的手指的方式抓持α-螺旋。截短和位点特异性突变显示，非结构化的N末端和连接β-链1和2的环对于胃蛋白酶抑制是重要的，但连接链3和4的环不是。使用基于诱变结果的模糊限制，然后将SQAPI计算对接到胃蛋白酶。所得模型将SQAPI的N-末端链置于底物结合裂缝的S'侧，而第一个SQAPI环结合在裂缝的S侧。SQAPI的骨架不与胃蛋白酶催化的Asp（32）-Asp（215）二联体相互作用，从而避免切割。数据显示SQAPI确实与半胱氨酸蛋白酶抑制剂共享同源结构元件，并且似乎保留了类似的蛋白酶抑制机制，尽管其靶标不同。这有力地支持了我们的假设，即SQAPI从祖先的半胱氨酸蛋白酶抑制剂进化而来。
The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NMR and show that SQAPI shares the same fold as a plant cystatin. The structure is characterized by a four-strand anti-parallel beta-sheet gripping an alpha-helix in an analogous manner to fingers of a hand gripping a tennis racquet. Truncation and site-specific mutagenesis revealed that the unstructured N terminus and the loop connecting beta-strands 1 and 2 are important for pepsin inhibition, but the loop connecting strands 3 and 4 is not. Using ambiguous restraints based on the mutagenesis results, SQAPI was then docked computationally to pepsin. The resulting model places the N-terminal strand of SQAPI in the S' side of the substrate binding cleft, whereas the first SQAPI loop binds on the S side of the cleft. The backbone of SQAPI does not interact with the pepsin catalytic Asp(32)-Asp(215) diad, thus avoiding cleavage. The data show that SQAPI does share homologous structural elements with cystatin and appears to retain a similar protease inhibitory mechanism despite its different target. This strongly supports our hypothesis that SQAPI evolved from an ancestral cystatin.