Cabrita Lisa D, Irving James A, Pearce Mary C, Whisstock James C, Bottomley Stephen P
Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia.
Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia; ARC Centre of Excellence for Structural and Function Microbial Genomics, Monash University, Clayton 3800, Australia.
J Biol Chem. 2007 Sep 14;282(37):26802-26809. doi: 10.1074/jbc.M705020200. Epub 2007 Jul 16.
Serpins are metastable proteinase inhibitors. Serpin metastability drives both a large conformational change that is utilized during proteinase inhibition and confers an inherent structural flexibility that renders serpins susceptible to aggregation under certain conditions. These include point mutations (the basis of a number of important human genetic diseases), small changes in pH, and an increase in temperature. Many studies of serpins from mesophilic organisms have highlighted an inverse relationship: mutations that confer a marked increase in serpin stability compromise inhibitory activity. Here we present the first biophysical characterization of a metastable serpin from a hyperthermophilic organism. Aeropin, from the archaeon Pyrobaculum aerophilum, is both highly stable and an efficient proteinase inhibitor. We also demonstrate that because of high kinetic barriers, aeropin does not readily form the partially unfolded precursor to serpin aggregation. We conclude that stability and activity are not mutually exclusive properties in the context of the serpin fold, and propose that the increased stability of aeropin is caused by an unfolding pathway that minimizes the formation of an aggregation-prone intermediate ensemble, thereby enabling aeropin to bypass the misfolding fate observed with other serpins.
丝氨酸蛋白酶抑制剂（Serpins）是亚稳态的蛋白酶抑制剂。丝氨酸蛋白酶抑制剂的亚稳态既驱动了蛋白酶抑制过程中发生的巨大构象变化，又赋予了固有的结构灵活性，使得丝氨酸蛋白酶抑制剂在某些条件下易于聚集。这些条件包括点突变（许多重要人类遗传疾病的基础）、pH值的微小变化以及温度升高。许多对嗜温生物来源的丝氨酸蛋白酶抑制剂的研究都强调了一种反比关系：赋予丝氨酸蛋白酶抑制剂稳定性显著增加的突变会损害其抑制活性。在此，我们展示了来自嗜热生物的亚稳态丝氨酸蛋白酶抑制剂的首次生物物理特性描述。来自嗜热栖热放线菌（Pyrobaculum aerophilum）的气单胞菌蛋白酶抑制剂（Aeropin）既高度稳定又是一种高效的蛋白酶抑制剂。我们还证明，由于存在高动力学屏障，气单胞菌蛋白酶抑制剂不容易形成丝氨酸蛋白酶抑制剂聚集的部分未折叠前体。我们得出结论，在丝氨酸蛋白酶抑制剂折叠的背景下，稳定性和活性并非相互排斥的特性，并提出气单胞菌蛋白酶抑制剂稳定性增加是由一种解折叠途径导致的，该途径使易于聚集的中间聚集体的形成最小化，从而使气单胞菌蛋白酶抑制剂能够绕过其他丝氨酸蛋白酶抑制剂所观察到的错误折叠命运。
