Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA.
Biophys J. 2009 Nov 18;97(10):2846-53. doi: 10.1016/j.bpj.2009.09.004.
Structures of truncated versions of the influenza A virus M2 proton channel have been determined recently by x-ray crystallography in the open conformation of the channel, and by NMR in the closed state. The structures differ in the position of the bound inhibitors. The x-ray structure shows a single amantadine molecule in the middle of the channel, whereas in the NMR structure four drug molecules bind at the channel's outer surface. To study this controversy we applied computational solvent mapping, a technique developed for the identification of the most druggable binding hot spots of proteins. The method moves molecular probes--small organic molecules containing various functional groups--around the protein surface, finds favorable positions using empirical free energy functions, clusters the conformations, and ranks the clusters on the basis of the average free energy. The results of the mapping show that in both structures the primary hot spot is an internal cavity overlapping the amantadine binding site seen in the x-ray structure. However, both structures also have weaker hot spots at the exterior locations that bind rimantadine in the NMR structure, although these sites are partially due to the favorable interactions with the interfacial region of the lipid bilayer. As confirmed by docking calculations, the open channel binds amantadine at the more favorable internal site, in good agreement with the x-ray structure. In contrast, the NMR structure is based on a peptide/micelle construct that is able to accommodate the small molecular probes used for the mapping, but has a too narrow pore for the rimantadine to access the internal hot spot, and hence the drug can bind only at the exterior sites.
最近，通过 X 射线晶体学在通道的开放构象中和通过 NMR 在关闭状态下，已经确定了流感 A 病毒 M2 质子通道的截断版本的结构。这些结构在结合抑制剂的位置上有所不同。X 射线结构显示在通道的中间有一个单一的金刚烷分子，而在 NMR 结构中，有四个药物分子结合在通道的外表面。为了研究这一争议，我们应用了计算溶剂映射技术，这是一种为蛋白质的最具成药性结合热点识别而开发的技术。该方法将分子探针（含有各种官能团的小分子）移动到蛋白质表面，使用经验自由能函数找到有利位置，对构象进行聚类，并根据平均自由能对聚类进行排名。映射结果表明，在这两种结构中，主要的热点是一个内部空腔，与 X 射线结构中看到的金刚烷结合位点重叠。然而，这两种结构在外层位置也有较弱的热点，在 NMR 结构中结合 rimantadine，尽管这些位点部分是由于与脂质双层的界面区域的有利相互作用。正如对接计算所证实的那样，开放通道在内部更有利的位置结合金刚烷，这与 X 射线结构一致。相比之下，NMR 结构基于一个肽/胶束构建体，能够容纳用于映射的小分子探针，但孔太窄，rimantadine 无法进入内部热点，因此药物只能结合在外层位置。
