Binding of Low Molecular Weight Inhibitors Promotes Large Conformational Changes in the Dengue Virus NS2B-NS3 Protease: Fold Analysis by Pseudocontact Shifts
Binding of Low Molecular Weight Inhibitors Promotes Large Conformational Changes in the Dengue Virus NS2B-NS3 Protease: Fold Analysis by Pseudocontact Shifts
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DOI：
10.1021/ja208435s
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发表时间：
2011-11-30
影响因子：
15
通讯作者：
Otting, Gottfried
中科院分区：
文献类型：
--
作者：
de la Cruz, Laura;Thi Hoang Duong Nguyen;Otting, Gottfried
双组分登革热病毒 NS2B-NS3 蛋白酶 (DEN NS2B-NS3pro) 是一个既定的药物靶点，但抑制剂设计因缺乏完全活性形式的蛋白酶的晶体结构而受到阻碍。在溶液中且没有抑制剂的情况下，NS2B 辅因子的功能重要的 C 端片段与 DEN NS3pro（“开放状态”）分离，需要进行较大的结构变化以产生被认为支持活性的“闭合状态”。我们通过核磁共振 (NMR) 光谱分析了含有和不含结合抑制剂的溶液中 DEN NS2B-NS3pro 的折叠。多个顺磁镧系元素标签被附着到不同的位点以产生伪接触位移（PCS）。面对严重的光谱重叠和构象交换导致的许多信号展宽，N-15-HSQC交叉峰的分配方法包括选择性突变、组合同位素标记以及比较实验PCS和使用相关西尼罗病毒（WNV）蛋白酶的结构作为模板构建的闭合构象结构模型反算的PCS。 PCS 表明，在带正电荷的低分子量抑制剂存在的情况下，该酶呈现关闭状态，这与之前观察到的 WNV 蛋白酶的关闭状态非常相似。因此，建立在 WNV 蛋白酶闭合构象上的蛋白酶模型是比现有晶体结构更好的合理药物设计模板，至少对于带正电荷的抑制剂而言是这样。为了评估开放状态，我们创建了 Gd3+ 复合物的结合位点并测量了顺磁弛豫增强。结果表明，DEN NS2B-NS3pro 晶体中显示的特定开放构象在溶液中几乎不存在。所使用的技术为对产生较差 NMR 谱的蛋白质进行折叠分析开辟了一条途径，因为来自多个位点的 PCS 与模型构建相结合，即使从不完全指定的 N-15-HSQC 谱中也能生成强大的信息。
The two-component dengue virus NS2B-NS3 protease (DEN NS2B-NS3pro) is an established drug target, but inhibitor design is hampered by the lack of a crystal structure of the protease in its fully active form. In solution and without inhibitors, the functionally important C-terminal segment of the NS2B cofactor is dissociated from DEN NS3pro ("open state"), necessitating a large structural change to produce the "closed state" thought to underpin activity. We analyzed the fold of DEN NS2B-NS3pro in solution with and without bound inhibitor by nuclear magnetic resonance (NMR) spectroscopy. Multiple paramagnetic lanthanide tags were attached to different sites to generate pseudocontact shifts (PCS). In the face of severe spectral overlap and broadening of many signals by conformational exchange, methods for assignment of N-15-HSQC cross-peaks included selective mutation, combinatorial isotope labeling, and comparison of experimental PCSs and PCSs back-calculated for a structural model of the closed conformation built by using the structure of the related West Nile virus (WNV) protease as a template. The PCSs show that, in the presence of a positively charged low-molecular weight inhibitor, the enzyme assumes a closed state that is very similar to the closed state previously observed for the WNV protease. Therefore, a model of the protease built on the closed conformation of the WNV protease is a better template for rational drug design than available crystal structures, at least for positively charged inhibitors. To assess the open state, we created a binding site for a Gd3+ complex and measured paramagnetic relaxation enhancements. The results show that the specific open conformation displayed in the crystal of DEN NS2B-NS3pro is barely populated in solution. The techniques used open an avenue to the fold analysis of proteins that yield poor NMR spectra, as PCSs from multiple sites in combination with model building generate powerful information even from incompletely assigned N-15-HSQC spectra.