Structural and mechanistic exploration of acid resistance: Kinetic stability facilitates evolution of extremophilic behavior
Structural and mechanistic exploration of acid resistance: Kinetic stability facilitates evolution of extremophilic behavior
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DOI：
10.1016/j.jmb.2007.02.032
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发表时间：
2007-05-04
影响因子：
5.6
通讯作者：
Agard, David A.
中科院分区：
文献类型：
--
作者：
Kelch, Brian A.;Eagen, Kyle P.;Agard, David A.
动力学稳定的蛋白质是独一无二的，因为它们的稳定性完全由动力学势垒决定，而不是由热力学平衡决定。为了更好地了解运动稳定性如何促进蛋白质在极端环境条件下的生存，我们分析了白色诺卡氏菌蛋白酶A(NAPase)的去折叠行为并确定了其结构，并将这些结果与中性粒细胞同源物α-裂解酶(α-LP)进行了比较。尽管NAPase和Alpha LP具有相同数量的酸可滴定残基，但动力学研究表明，NAPase展开自由能垒的高度对酸的敏感性低于Alpha LP，从而解释了NAPase对低pH的耐受性提高。对ALP和NAPase结构的比较发现，在(αLP)的结构域界面上有多个盐桥被重新定位到NAPase的外部区域，这表明了一种新的酸稳定机制，在这种机制中，酸敏感的静电相互作用被重排，以类似地影响本征状态和展开过渡态的能量。(αLP的一个酸稳定变体，其中单个结构域间盐桥被相应的结构域内NAPase盐桥取代，显示出耐酸性显著增加15倍，为这一假说提供了进一步的证据。这些观察也导致了过渡态结构彼此展开而本身保持相对完整的一般模型。这些结果表明，运动稳定性作为一种进化工具，在广泛的恶劣条件下发展寿命具有显着的实用性。(C)2007爱思唯尔有限公司。保留所有权利。
Kinetically stable proteins are unique in that their stability is determined solely by kinetic barriers rather than by thermodynamic equilibria. To better understand how kinetic stability promotes protein survival under extreme environmental conditions, we analyzed the unfolding behavior and determined the structure of Nocardiopsis alba Protease A (NAPase), an acid-resistant, kinetically stable protease, and compared these results with a neutrophilic homolog, alpha-lytic protease (alpha LP). Although NAPase and alpha LP have the same number of acid-titratable residues, kinetic studies revealed that the height of the unfolding free energy barrier for NAPase is less sensitive to acid than that of (alpha LP, thereby accounting for NAPase's improved tolerance of low pH. A comparison of the aLP and NAPase structures identified multiple salt-bridges in the domain interface of (alpha LP that were relocated to outer regions of NAPase, suggesting a novel mechanism of acid stability in which acid-sensitive electrostatic interactions are rearranged to similarly affect the energetics of both the native state and the unfolding transition state. An acid-stable variant of (alpha LP in which a single interdomain salt-bridge is replaced with a corresponding intradomain NAPase salt-bridge shows a dramatic > 15-fold increase in acid resistance, providing further evidence for this hypothesis. These observations also led to a general model of the unfolding transition state structure each other while remaining relatively intact themselves. These results illustrate the remarkable utility of kinetic stability as an evolutionary tool for developing longevity over a broad range of harsh conditions. (c) 2007 Elsevier Ltd. All rights reserved.