Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
J Am Chem Soc. 2012 Mar 7;134(9):4163-8. doi: 10.1021/ja2095766. Epub 2012 Feb 28.
Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.
人类免疫缺陷病毒 1 型（HIV-1）蛋白酶对病毒成熟和感染性至关重要。对蛋白酶动力学的研究表明，疏水区的重排对于酶活性是必需的。疏水区的许多突变也与耐药性相关，并可能调节核心的柔韧性。为了测试柔韧性在蛋白酶活性中的作用，在远离活性位点的柔性区域的界面处引入了一对半胱氨酸。通过晶体结构和对游离半胱氨酸的烷基化和质谱分析证实了二硫键的形成。这些变体的氧化和还原晶体结构表明蛋白酶的整体结构得以保留。然而，半胱氨酸的交联导致酶活性急剧丧失，而当二硫键交联减少时，酶活性又得以恢复。分子动力学模拟表明，从工程化的二硫键中，改变的动力学在整个酶中传播。因此，疏水区内柔韧性的改变可以调节 HIV-1 蛋白酶的活性，支持这样一种假设，即远离活性位点的耐药性突变可以通过调节酶活性来改变底物周转和抑制剂结合之间的平衡。
