Sicoli Giuseppe, Konijnenberg Albert, Guérin Jérémy, Hessmann Steve, Del Nero Elise, Hernandez-Alba Oscar, Lecher Sophie, Rouaut Guillaume, Müggenburg Linn, Vezin Hervé, Cianférani Sarah, Sobott Frank, Schneider Robert, Jacob-Dubuisson Françoise
Laboratoire Avancé de Spectroscopie pour les Interactions, la Réactivité et l'Environnement (LASIRE), UMR CNRS 8516, Université de Lille, Lille, France.
BAMS Research Group, University of Antwerp, Antwerp, Belgium.
Front Mol Biosci. 2022 Jul 22;9:950871. doi: 10.3389/fmolb.2022.950871. eCollection 2022.
The Two-Partner secretion pathway mediates protein transport across the outer membrane of Gram-negative bacteria. TpsB transporters belong to the Omp85 superfamily, whose members catalyze protein insertion into, or translocation across membranes without external energy sources. They are composed of a transmembrane β barrel preceded by two periplasmic POTRA domains that bind the incoming protein substrate. Here we used an integrative approach combining assays, mass spectrometry, nuclear magnetic resonance and electron paramagnetic resonance techniques suitable to detect minor states in heterogeneous populations, to explore transient conformers of the TpsB transporter FhaC. This revealed substantial, spontaneous conformational changes on a slow time scale, with parts of the POTRA2 domain approaching the lipid bilayer and the protein's surface loops. Specifically, our data indicate that an amphipathic POTRA2 β hairpin can insert into the β barrel. We propose that these motions enlarge the channel and initiate substrate secretion. Our data propose a solution to the conundrum how TpsB transporters mediate protein secretion without the need for cofactors, by utilizing intrinsic protein dynamics.
双伙伴分泌途径介导革兰氏阴性菌外膜蛋白的转运。TpsB转运蛋白属于Omp85超家族，其成员可在无外部能量来源的情况下催化蛋白质插入膜或跨膜转运。它们由一个跨膜β桶结构域和两个周质POTRA结构域组成，后者可结合进入的蛋白质底物。在这里，我们采用了一种综合方法，结合了适合检测异质群体中微小状态的分析、质谱、核磁共振和电子顺磁共振技术，以探索TpsB转运蛋白FhaC的瞬时构象。这揭示了在较慢时间尺度上发生的大量自发构象变化，POTRA2结构域的部分区域靠近脂质双层和蛋白质的表面环。具体而言，我们的数据表明，一个两亲性的POTRA2β发夹可以插入β桶结构中。我们提出，这些运动扩大了通道并启动了底物分泌。我们的数据提出了一个难题的解决方案，即TpsB转运蛋白如何通过利用内在蛋白质动力学在无需辅因子的情况下介导蛋白质分泌。
