The structural stability of the HIV-1 protease.
The structural stability of the HIV-1 protease.
复制标题
DOI：
10.1006/jmbi.1998.2090
复制
发表时间：
1998-10
影响因子：
5.6
通讯作者：
M. Todd;N. Semo;E. Freire
中科院分区：
文献类型：
--
作者：
M. Todd;N. Semo;E. Freire
HIV-1 蛋白酶抑制剂开发中最常见的策略是设计高亲和力过渡态类似物，有效与天然底物竞争活性位点。第二种方法是开发通过破坏蛋白酶四级或三级结构的稳定性来灭活蛋白酶的化合物。这些策略的成功优化需要准确了解结构稳定和结合的能量学，并识别蛋白酶分子中对稳定性和功能至关重要的区域。在这里，首次通过高灵敏度差示扫描量热法测量了 HIV-1 蛋白酶的稳定能量。这些研究允许评估吉布斯稳定能的不同组成部分（焓、熵和热容变化）。蛋白酶的稳定性取决于 pH 值，并且由于其二聚体性质，也取决于浓度。在 pH 3.4 时，25 ℃ 时吉布斯稳定能接近 10 kcal/mol，与 5x10(-8) M 的解离常数一致。蛋白酶的稳定性在较高 pH 值下增加。在 pH 5 时，25°C 时的吉布斯稳定能为 14.5 kcal/mol，与 2.3x10(-11) M 的解离常数一致。稳定吉布斯能的 pH 依赖性表明，在 pH 3.4 和 pH 5 之间，每个二聚体平均有 3-4 个可电离基团在展开时质子化。对蛋白酶分子进行基于结构的热力学分析表明，大部分吉布斯稳定能是由二聚化界面提供的，并且分离的亚基本质上不稳定。然而，吉布斯能量沿二聚界面并不均匀分布。二聚体界面的特征是存在对亚基关联有显着贡献的残基簇（热点）和对亚基关联贡献很小的其他区域。在二聚化界面，位于羧基和氨基末端的残基贡献了接近 75% 的总吉布斯能量（Cys95、Thr96、Leu97、Asn98 和 Phe99 以及 Pro1、Ile3、Leu5）。位于活性位点底部的残基 Thr26、Gly27 和 Asp29 也很重要，并且在较小程度上位于皮瓣区域尖端的残基 Gly49、Ile50、Gly51。基于结构的热力学分析还预测蛋白酶区域的存在，该区域仅具有边际稳定性并且具有高度独立局部解折叠的倾向。特别是，皮瓣区域占据非常浅的能量最小值，并且其构象很容易受到相对较小的扰动的影响。蛋白酶的这种特性可能与某些突变引发对某些抑制剂的抗性的能力有关。
The most common strategy in the development of HIV-1 protease inhibitors has been the design of high affinity transition state analogs that effectively compete with natural substrates for the active site. A second approach has been the development of compounds that inactivate the protease by destabilizing its quaternary or tertiary structure. A successful optimization of these strategies requires an accurate knowledge of the energetics of structural stabilization and binding, and the identification of those regions in the protease molecule that are critical to stability and function. Here the energetics of stabilization of the HIV-1 protease has been measured for the first time by high sensitivity differential scanning calorimetry. These studies have permitted the evaluation of the different components of the Gibbs energy of stabilization (the enthalpy, entropy and heat capacity changes). The stability of the protease is pH-dependent and due to its dimeric nature is also concentration-dependent. At pH 3.4 the Gibbs energy of stabilization is close to 10 kcal/mol at 25 degreesC, consistent with a dissociation constant of 5x10(-8) M. The stability of the protease increases at higher pH values. At pH 5, the Gibbs energy of stabilization is 14.5 kcal/mol at 25 degreesC, consistent with a dissociation constant of 2.3x10(-11) M. The pH dependence of the Gibbs energy of stabilization indicates that between pH 3.4 and pH 5 an average of 3-4 ionizable groups per dimer become protonated upon unfolding. A structure-based thermodynamic analysis of the protease molecule indicates that most of the Gibbs energy of stabilization is provided by the dimerization interface and that the isolated subunits are intrinsically unstable. The Gibbs energy, however, is not uniformly distributed along the dimerization interface. The dimer interface is characterized by the presence of clusters of residues (hot spots) that contribute significantly and other regions that contribute very little to subunit association. At the dimerization interface, residues located at the carboxy and amino termini contribute close to 75% of the total Gibbs energy (Cys95, Thr96, Leu97, Asn98 and Phe99 and Pro1, Ile3, Leu5). Residues Thr26, Gly27 and Asp29 located at the base of the active site are also important, and to a lesser extent Gly49, Ile50, Gly51 located at the tip of the flap region. The structure-based thermodynamic analysis also predicts the existence of regions of the protease with only marginal stability and a high propensity to undergo independent local unfolding. In particular, the flap region occupies a very shallow energy minimum and its conformation can easily be affected by relatively small perturbations. This property of the protease can be related to the ability of some mutations to elicit resistance towards certain inhibitors.