Kawamura Takahiro, Wakamoto Takuro, Kitazawa Soichiro, Sakuraba Shun, Kameda Tomoshi, Kitahara Ryo
College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga, Japan.
College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Shiga, Japan.
Biophys J. 2017 May 9;112(9):1820-1828. doi: 10.1016/j.bpj.2017.03.029.
Internal cavities in proteins produce conformational fluctuations and enable the binding of small ligands. Here, we report a NMR analysis of O-binding sites by O-induced paramagnetic relaxation enhancements (PREs) on amide groups of proteins in solution. Outer surface protein A contains a nonglobular single-layer β-sheet that connects the N- and C-terminal globular domains. Several cavities have been observed in both domains of the crystallized protein structure. The receptor-binding sites are occluded and line the largest cavity of the C-terminal domain. We observed significant O-induced PREs for amide protons located around the largest cavity and at the central β-sheet. We suggested three potential O-accessible sites in the protein based on the 1/r distance dependence of the PRE. Two sites were in or close to the largest cavity and the third site was in the surface crevice of the central β-sheet. These results provide, to our knowledge, the first evidence of ligand binding to the surface crevice and cavity of the protein in solution. Because O generally binds more specifically to hydrophobic rather than hydrophilic cavities within a protein, the results also indicated that the receptor-binding sites lining the largest cavity were in the hydrophobic environment in the ground-state conformation. Molecular dynamics simulations permitted the visualization of the rotational and translational motions of O within the largest cavity, egress of O from the cavity, and ingress of O in the surface crevice of the β-sheet. These molecular dynamics simulation results qualitatively explained the O-induced changes in NMR observations. Exploring cavities that are sufficiently dynamic to enable access by small molecules can be a useful strategy for the design of stable proteins and their ligands.
蛋白质内部的腔体会产生构象波动，并有助于小分子配体的结合。在此，我们报告了通过溶液中蛋白质酰胺基团上的氧诱导顺磁弛豫增强（PREs）对氧结合位点进行的核磁共振分析。外表面蛋白A包含一个非球状的单层β折叠，连接N端和C端球状结构域。在结晶蛋白结构的两个结构域中都观察到了几个腔体。受体结合位点被封闭，位于C端结构域的最大腔体周围。我们观察到位于最大腔体周围和中央β折叠处的酰胺质子有显著的氧诱导PREs。基于PRE的1/r距离依赖性，我们在该蛋白中提出了三个潜在的氧可及位点。两个位点位于最大腔体内部或附近，第三个位点位于中央β折叠的表面裂隙中。据我们所知，这些结果首次证明了配体在溶液中与蛋白的表面裂隙和腔体结合。由于氧通常更特异性地结合到蛋白内部的疏水而非亲水腔体中，这些结果还表明，位于最大腔体周围的受体结合位点处于基态构象的疏水环境中。分子动力学模拟使我们能够可视化氧在最大腔体中的旋转和平移运动、氧从腔体中逸出以及氧进入β折叠的表面裂隙。这些分子动力学模拟结果定性地解释了氧诱导的核磁共振观测变化。探索具有足够动态性以允许小分子进入的腔体，可能是设计稳定蛋白质及其配体的一种有用策略。
