Department of Chemistry , University of Massachusetts , 374 LGRT, 710 North Pleasant Street , Amherst , Massachusetts 01003 , United States.
Biochemistry. 2019 Feb 12;58(6):776-787. doi: 10.1021/acs.biochem.8b00978. Epub 2018 Dec 17.
The dengue virus protease (NS2B-NS3pro) plays a critical role in the dengue viral life cycle, making it an attractive drug target for dengue-related pathologies, including dengue hemorrhagic fever. A number of studies indicate that NS2B-NS3pro undergoes a transition between two widely different conformational states: an "open" (inactive) conformation and a "closed" (active) conformation. For the past several years, the equilibrium between these states and the resting conformation of NS2B-NS3pro have been debated, although a strong consensus is emerging. To investigate the importance of such conformational states, we developed versions of NS2B-NS3pro that allow us to trap the enzyme in various distinct conformations. Our data from these variants suggest that the enzymatic activity appears to be dependent on the movement of NS2B and may rely on the flexibility of the protease core. Locking the enzyme into the "closed" conformation dramatically increased activity, strongly suggesting that the "closed" conformation is the active conformation. The observed resting state of the enzyme depends largely on the construct used to express the NS2B-NS3pro complex. In an "unlinked" construct, in which the NS2B and NS3 regions exist as independent, co-expressed polypeptides, the enzyme rests predominantly in a "closed", active conformation. In contrast, in a "linked" construct, in which NS2B and NS3 are attached by a nine-amino acid linker, NS2B-NS3pro adopts a more relaxed, alternative conformation. Nevertheless, even the unlinked construct samples both the "closed" and other alternative conformations. Given our findings, and the more realistic resemblance of NS2B-NS3pro to the native enzyme, these data strongly suggest that studies should focus on the "unlinked" constructs moving forward. Additionally, the results from these studies provide a more detailed understanding of the various poses of the dengue virus NS2B-NS3 protease and should help guide future drug discovery efforts aimed at this enzyme.
登革热病毒蛋白酶（NS2B-NS3pro）在登革热病毒生命周期中起着至关重要的作用，使其成为与登革热相关病理相关的有吸引力的药物靶点，包括登革出血热。许多研究表明，NS2B-NS3pro 经历了两种广泛不同构象状态之间的转变：“开放”（非活性）构象和“闭合”（活性）构象。在过去的几年中，这些状态之间的平衡和 NS2B-NS3pro 的静止构象一直存在争议，尽管正在形成强烈的共识。为了研究这些构象状态的重要性，我们开发了可以将酶固定在各种不同构象中的 NS2B-NS3pro 版本。我们从这些变体中获得的数据表明，酶的活性似乎取决于 NS2B 的运动，并且可能依赖于蛋白酶核心的灵活性。将酶锁定在“闭合”构象中会极大地增加活性，强烈表明“闭合”构象是活性构象。酶的观察到的静止状态在很大程度上取决于用于表达 NS2B-NS3pro 复合物的构建体。在“未连接”构建体中，其中 NS2B 和 NS3 区域作为独立的、共表达的多肽存在，酶主要处于“闭合”、活性构象。相比之下，在“连接”构建体中，其中 NS2B 和 NS3 通过九个氨基酸的接头连接，NS2B-NS3pro 采用更松弛的替代构象。然而，即使是未连接的构建体也会同时采样“闭合”和其他替代构象。考虑到我们的发现，以及 NS2B-NS3pro 与天然酶更相似，这些数据强烈表明，未来的研究应集中在“未连接”构建体上。此外，这些研究的结果提供了对登革热病毒 NS2B-NS3 蛋白酶的各种构象的更详细的了解，应该有助于指导针对该酶的未来药物发现工作。
