Structural basis for oligomerization of the prokaryotic peptide transporter PepTSo2
Structural basis for oligomerization of the prokaryotic peptide transporter PepTSo2
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DOI：
10.1107/s2053230x19003546
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发表时间：
2019-05-01
影响因子：
0.9
通讯作者：
Nureki, Osamu
中科院分区：
文献类型：
--
作者：
Nagamura, Reina;Fukuda, Masahiro;Nureki, Osamu
质子依赖性寡肽转运蛋白（POTs）属于主要易化剂超家族（MFS），将二肽和三肽从细胞外环境转运到靶细胞。人POTs PepT 1和PepT 2也参与各种口服摄入药物的吸收。先前报道的结构显示细菌POT具有14个螺旋，其中H1-H6和H7-H12构成典型的MFS折叠，剩余的两个螺旋参与胞质接头。来自Shewanella oneidensis的PepT（So2）是一种独特的POT，据报道其组装为200 kDa四聚体。虽然先前报道的结构表明H12对四聚体形成的重要性，但由于缺乏高分辨率的四聚体结构，PepT（So2）特异性寡聚化的结构基础仍然不清楚。本研究优化了四聚体PepT（So2）的表达和纯化条件。单颗粒冷冻-EM分析揭示了以4.1埃分辨率并入Salipro纳米颗粒中的PepT（So2）的四聚体结构。此外，一个组合的无规立方相（LCP）结晶和多个微晶的自动化数据处理系统，使PepT（SO 2）的晶体结构被确定在3.5和3.9埃的分辨率。在脂质双层中的现有结构揭示了PepT（So2）的四聚体组装的详细机制，其中特征性的细胞外环（ECL）与H12上的两个天冬酰胺残基相互作用，据报道这对于四聚体是重要的，并且在寡聚体组装中起着至关重要的作用。这项研究提供了有价值的见解，这种MFS型转运蛋白的寡聚化机制，这将进一步铺平道路，了解其他寡聚膜蛋白。
Proton-dependent oligopeptide transporters (POTs) belong to the major facilitator superfamily (MFS) and transport dipeptides and tripeptides from the extracellular environment into the target cell. The human POTs PepT1 and PepT2 are also involved in the absorption of various orally ingested drugs. Previously reported structures revealed that the bacterial POTs possess 14 helices, of which H1-H6 and H7-H12 constitute the typical MFS fold and the residual two helices are involved in the cytoplasmic linker. PepT(So2) from Shewanella oneidensis is a unique POT which reportedly assembles as a 200 kDa tetramer. Although the previously reported structures suggested the importance of H12 for tetramer formation, the structural basis for the PepT(So2)-specific oligomerization remains unclear owing to the lack of a high-resolution tetrameric structure. In this study, the expression and purification conditions for tetrameric PepT(So2) were optimized. A single-particle cryo-EM analysis revealed the tetrameric structure of PepT(So2) incorporated into Salipro nanoparticles at 4.1 angstrom resolution. Furthermore, a combination of lipidic cubic phase (LCP) crystallization and an automated data-processing system for multiple microcrystals enabled crystal structures of PepT(So2) to be determined at resolutions of 3.5 and 3.9 angstrom. The present structures in a lipid bilayer revealed the detailed mechanism for the tetrameric assembly of PepT(So2), in which a characteristic extracellular loop (ECL) interacts with two asparagine residues on H12 which were reported to be important for tetramerization and plays an essential role in oligomeric assembly. This study provides valuable insights into the oligomerization mechanism of this MFS-type transporter, which will further pave the way for understanding other oligomeric membrane proteins.