Müller Reinke T, Travers Timothy, Cha Hi-Jea, Phillips Joshua L, Gnanakaran S, Pos Klaas M
Institute of Biochemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany.
Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM 87545, United States; Center for Nonlinear Sciences, Los Alamos National Laboratory, Los Alamos, NM 87545, United States.
J Mol Biol. 2017 Dec 8;429(24):3863-3874. doi: 10.1016/j.jmb.2017.09.018. Epub 2017 Oct 5.
The functionally important switch loop of the trimeric multidrug transporter AcrB separates the access and deep drug binding pockets in every protomer. This loop, comprising 11-amino-acid residues, has been shown to be crucial for substrate transport, as drugs have to travel past the loop to reach the deep binding pocket and from there are transported outside the cell via the connected AcrA and TolC channels. It contains four symmetrically arranged glycine residues suggesting that flexibility is a key feature for pump activity. Upon combinatorial substitution of these glycine residues to proline, functional and structural asymmetry was observed. Proline substitutions on the PC1-proximal side completely abolished transport and reduced backbone flexibility of the switch loop, which adopted a conformation restricting the pathway toward the deep binding pocket. Two phenylalanine residues located adjacent to the substitution sensitive glycine residues play a role in blocking the pathway upon rigidification of the loop, since the removal of the phenyl rings from the rigid loop restores drug transport activity.
三聚体多药转运蛋白AcrB的功能重要开关环在每个原体中分隔了入口和深部药物结合口袋。这个由11个氨基酸残基组成的环已被证明对底物转运至关重要，因为药物必须经过该环才能到达深部结合口袋，然后通过相连的AcrA和TolC通道被转运出细胞。它包含四个对称排列的甘氨酸残基，这表明灵活性是泵活性的关键特征。在将这些甘氨酸残基组合替换为脯氨酸后，观察到了功能和结构的不对称性。PC1近端一侧的脯氨酸替换完全消除了转运，并降低了开关环的主链灵活性，该环采用了一种限制通往深部结合口袋途径的构象。位于对替换敏感的甘氨酸残基相邻位置的两个苯丙氨酸残基在环刚性化时起到了阻断途径的作用，因为从刚性环上去除苯环可恢复药物转运活性。
