P1 – Comprehensive structural description of SLC26 membrane transport and its modulation

细胞生理学关键取决于跨膜氯离子和碳酸氢根离子梯度的产生和维持。来自SLC 26家族的溶质载体在此过程中发挥重要作用，如其在某些病理学中的致病作用所示。最近的结构揭示了整体蛋白质的结构，并揭示了一个独特的安排，包括交换安排的细胞质STAS结构域的二聚体中的原聚体。进一步的功能分析强调了STAS结构域在加速运输中的作用，并证明了二聚体中原聚体之间的合作相互作用。然而，由于现有的结构没有描述一个完整的易位循环，它仍然不清楚如何运输，其调制，以及原聚体之间的协同性。我们的目标是为解决这些问题提供一个广泛的结构和机制框架。我们将首先描绘完整的构象空间的哺乳动物SLC 26转运蛋白在脂质环境中使用一种独特的方法，涉及：（i）小抗体，逮捕离散构象的蛋白质;和（ii）单粒子冷冻EM结构测定。具有相反构象的原聚体的转运蛋白的后续结构将揭示SLC 26二聚体中功能协同性的基础。最后，涉及STAS结构域的二聚体接口的结构和功能的询问将导致STAS介导的运输调制的机械赞赏。再加上研究单位内的互补合作，解决进一步的功能和动态方面，我们希望在SLC 26运输提供前所未有的机械见解。

Cellular physiology critically depends on the generation and maintenance of chloride and bicarbonate ion gradients across membranes. Solute carriers from the SLC26 family play an important role in this process as illustrated by their causative role in certain pathologies. Recent structures uncovered the overall protein architecture and revealed a unique arrangement of protomers in the dimer including a swapped arrangement of the cytoplasmic STAS domain. Further functional analysis highlighted a role of the STAS domain in accelerating transport, and demonstrated cooperative interactions between the protomers in the dimer. However, as the available structures do not describe a complete translocation cycle, it has remained unclear how transport, its modulation, and the cooperativity between protomers are integrated. We aim to provide an extensive structural and mechanistic framework for addressing these questions. We will first delineate the complete conformation space of mammalian SLC26 transporters in a lipid environment using a unique methodological approach involving: (i) small antibodies, to arrest the proteins in discrete conformations; and (ii) single-particle cryo-EM for structure determination. Subsequent structures of transporters with protomers in opposite conformations will uncover the basis for functional cooperativity in the SLC26 dimer. Finally, structural and functional interrogations of the dimer interfaces involving the STAS domain will result in a mechanistic appreciation of STAS-mediated modulation of transport. Together with complementary collaborations within the Research Unit addressing further functional and dynamic aspects, we expect to provide unprecedented mechanistic insights in SLC26 transport.