Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
J Am Chem Soc. 2011 Nov 30;133(47):19205-15. doi: 10.1021/ja208435s. Epub 2011 Nov 9.
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 (15)N-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 Gd(3+) 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 (15)N-HSQC spectra.
二组分登革热病毒 NS2B-NS3 蛋白酶（DEN NS2B-NS3pro）是一个既定的药物靶点，但由于缺乏该蛋白酶完全活性形式的晶体结构，抑制剂的设计受到阻碍。在溶液中且没有抑制剂的情况下，功能上重要的 NS2B 辅助因子的 C 末端片段与 DEN NS3pro 解离（“开放状态”），需要进行很大的结构改变才能产生被认为是活性基础的“封闭状态”。我们通过核磁共振（NMR）光谱分析了结合和未结合抑制剂时 DEN NS2B-NS3pro 在溶液中的构象。将多个顺磁镧系元素标签连接到不同的位点以产生赝接触位移（PCS）。在面对严重的光谱重叠和构象交换引起的许多信号展宽的情况下，（15）N-HSQC 交叉峰的归属方法包括选择性突变、组合同位素标记以及比较实验 PCS 和根据封闭构象的结构模型计算的 PCS，该模型是使用相关的西尼罗河病毒（WNV）蛋白酶的结构作为模板构建的。PCS 表明，在带正电荷的低分子量抑制剂存在下，该酶呈现出非常类似于先前观察到的 WNV 蛋白酶的封闭状态。因此，基于 WNV 蛋白酶的封闭构象构建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是如此。为了评估开放状态，我们创建了一个与 Gd(3+) 配合物结合的结合位点并测量了顺磁弛豫增强。结果表明，在 DEN NS2B-NS3pro 晶体中显示的特定开放构象在溶液中几乎没有存在。这些技术为那些产生不良 NMR 谱的蛋白质的构象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，即使在未完全归属的（15）N-HSQC 谱中也能产生强大的信息。
