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

细胞生理学主要依赖于氯离子和碳酸氢盐离子在膜上梯度的产生和维持。SLC26家族的溶质载体在这一过程中发挥了重要作用，它们在某些病理中起着致病作用。最近的结构揭示了整个蛋白质结构，并揭示了二聚体中独特的原蛋白排列，包括细胞质STAS结构域的交换排列。进一步的功能分析强调了STAS结构域在加速转运中的作用，并证明了二聚体中原聚体之间的合作相互作用。然而，由于现有的结构并不能描述一个完整的易位周期，因此目前尚不清楚转运、其调制和原聚体之间的协同性是如何整合的。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。我们将首先使用一种独特的方法来描绘哺乳动物SLC26转运体在脂质环境中的完整构象空间，其中包括：(i)小抗体，以捕获离散构象中的蛋白质；（ii）单颗粒冷冻电镜（cryo-EM）用于结构测定。具有相反构象的转运蛋白的后续结构将揭示SLC26二聚体功能协同性的基础。最后，对涉及STAS结构域的二聚体界面的结构和功能分析将导致对STAS介导的转运调制的机制欣赏。加上研究部门内部的互补合作，进一步解决功能和动态方面的问题，我们期望在SLC26运输中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.