Osten Veronika, Oepen Kristin, Schneider Dirk
Department of Chemistry - Biochemistry, Johannes Gutenberg-University, Mainz, Germany.
Department of Chemistry - Biochemistry, Johannes Gutenberg-University, Mainz, Germany; Institute of Molecular Physiology, Johannes Gutenberg-University, Mainz, Germany.
J Biol Chem. 2025 Feb;301(2):108098. doi: 10.1016/j.jbc.2024.108098. Epub 2024 Dec 18.
ABC transporters are membrane integral proteins that consist of a transmembrane domain and nucleotide-binding domain (NBD). Two monomers (half-transporters) of the Bacillus subtilis ABC transporter Bacillus multidrug-resistance ATP (BmrA) dimerize to build a functional full-transporter. As all ABC exporters, BmrA uses the free energy of ATP hydrolysis to transport substrate molecules across the cell membrane. For substrate transport, a BmrA dimer undergoes major conformational changes. ATP binding drives dimerization of the NBDs followed by the hydrolysis of the nucleotides. Conserved structural elements within the NBD and transmembrane domain are crucial for dimerization and the activity of BmrA. In the BmrA structure, an α-helix is present at the C-terminus, which can be subdivided in two smaller helices. As shown here, the very C-terminal helix (fragment) is not crucial for the BmrA activity. In fact, based on Cys-scanning mutagenesis, this region is highly flexible. In contrast, a BmrA variant lacking the entire C-terminal α-helix, showed no ATPase and transport activity. Via Ala-scanning, we identified residues in the N-terminal fragment of the helix that are crucial for the BmrA activity, most likely via establishing contacts to structural elements involved in ATP recognition, binding, and/or hydrolysis.
ABC转运蛋白是由跨膜结构域和核苷酸结合结构域（NBD）组成的膜整合蛋白。枯草芽孢杆菌ABC转运蛋白多药耐药ATP（BmrA）的两个单体（半转运体）二聚化形成一个功能性的全转运体。与所有ABC外排泵一样，BmrA利用ATP水解的自由能将底物分子转运穿过细胞膜。对于底物转运，BmrA二聚体会发生重大构象变化。ATP结合驱动NBDs二聚化，随后核苷酸水解。NBD和跨膜结构域内保守的结构元件对于BmrA的二聚化和活性至关重要。在BmrA结构中，C末端存在一个α螺旋，可细分为两个较小的螺旋。如此处所示，最末端的C螺旋（片段）对BmrA活性并不关键。事实上，基于半胱氨酸扫描诱变，该区域具有高度灵活性。相比之下，一个缺失整个C末端α螺旋的BmrA变体则没有ATP酶活性和转运活性。通过丙氨酸扫描，我们确定了该螺旋N末端片段中的一些残基对BmrA活性至关重要，最有可能是通过与参与ATP识别、结合和/或水解的结构元件建立联系来实现的。
