NMR-Supported Structural Biology, Leibniz-Institut für Molekulare Pharmakologie, R.-Rössle-Strasse 10, 13125 Berlin, Germany.
Chembiochem. 2010 Mar 1;11(4):547-55. doi: 10.1002/cbic.200900472.
ATP-binding cassette (ABC) transport systems facilitate the translocation of substances, like amino acids, across cell membranes energised by ATP hydrolysis. This work describes first structural studies on the ABC transporter ArtMP from Geobacillus stearothermophilus in native lipid environment by magic-angle spinning NMR spectroscopy. The 2D crystals of ArtMP and 3D crystals of isolated ArtP were prepared in different nucleotide-bound or -unbound states. From selectively (13)C,(15)N-labelled ArtP, several sequence-specific assignments were obtained, most of which could be transferred to spectra of ArtMP. Residues Tyr133 and Pro134 protrude directly into the ATP-binding pocket at the interface of the ArtP subunits, and hence, are sensitive monitors for structural changes during nucleotide binding and hydrolysis. Distinct sets of NMR shifts were obtained for ArtP with different phosphorylation states of the ligand. Indications were found for an asymmetric or inhomogeneous state of the ArtP dimer bound with triphosphorylated nucleotides. With this investigation, a model system was established for screening all functional states occurring in one ABC transporter in native lipid environment.
ATP 结合盒（ABC）转运蛋白系统通过利用 ATP 水解产生的能量促进物质（如氨基酸）穿过细胞膜的转运。这项工作首次通过魔角旋转 NMR 光谱学研究了嗜热脂肪地芽孢杆菌的 ABC 转运蛋白 ArtMP 在天然脂质环境中的结构。ArtMP 的 2D 晶体和分离的 ArtP 的 3D 晶体是在不同核苷酸结合或非结合状态下制备的。从选择性（13）C，（15）N 标记的 ArtP 中获得了几个序列特异性分配，其中大多数可以转移到 ArtMP 的光谱中。残基 Tyr133 和 Pro134 直接突入 ArtP 亚基之间的 ATP 结合口袋，因此是监测核苷酸结合和水解过程中结构变化的敏感探针。对于具有不同配体磷酸化状态的 ArtP，获得了不同的 NMR 位移集。表明与三磷酸化核苷酸结合的 ArtP 二聚体处于不对称或不均匀状态。通过这项研究，在天然脂质环境中建立了一个筛选 ABC 转运蛋白中所有功能状态的模型系统。
