Protein-protein interactions in AQP regulation - biophysical characterization of AQP0-CaM and AQP2-LIP5 complex formation
Protein-protein interactions in AQP regulation - biophysical characterization of AQP0-CaM and AQP2-LIP5 complex formation
复制标题
DOI：
10.1039/c8fd00065d
复制
发表时间：
2018-09-01
影响因子：
3.4
通讯作者：
Toernroth-Horsefield, Susanna
中科院分区：
文献类型：
--
作者：
Kreida, Stefan;Roche, Jennifer Virginia;Toernroth-Horsefield, Susanna
蛋白-蛋白相互作用在调节人体水通道蛋白（AQP）的门控和转运中起重要作用。虽然结构和功能研究已经提供了AQP转运机制、选择性以及环或末端构象变化的门控的详细知识，但蛋白质-蛋白质相互作用如何控制AQP介导的水通过细胞膜转运的机制仍然缺乏表征。本文探讨了两种人类aqp与调节蛋白之间的相互作用：AQP0与钙调蛋白之间的相互作用，AQP0与钙调蛋白之间的相互作用介导AQP0的门控，以及AQP2与参与转运的LIP5之间的相互作用。利用微尺度热泳（MST）和荧光各向异性这两种具有低样品消耗和洗涤剂相容性的方法，我们发现可以使用全长AQP和与调节蛋白结合位点对应的AQP肽来研究相互作用。然而，全长aqp在不同的方法之间具有更好的再现性，并且首次揭示了AQP0以合作的方式与CaM结合，这在多肽实验中是没有发现的。我们的研究强调，虽然肽是定位结合位点和精确定位相互作用残基的重要工具，但全长蛋白可能提供额外的见解，如结合机制、变构和协同性，这是理解细胞背景下蛋白-蛋白介导调节的重要参数。我们的工作为AQP调控的进一步研究提供了一个平台，这可能对设计针对AQP复合物的药物以及开发用于水净化目的的人工仿生水通道感兴趣。
Protein-protein interactions play important roles in regulating human aquaporins (AQP) by gating as well as trafficking. While structural and functional studies have provided detailed knowledge of AQP transport mechanisms, selectivity as well as gating by conformational changes of loops or termini, the mechanism behind how protein-protein interactions control AQP-mediated water transport through cellular membranes remains poorly characterized. Here we explore the interaction between two human AQPs and regulatory proteins: the interaction between AQP0 and calmodulin, which mediates AQP0 gating, as well as the interaction between AQP2 and LIP5, which is involved in trafficking. Using microscale thermophoresis (MST) and fluorescence anisotropy, two methods that have the advantage of low sample consumption and detergent compatibility, we show that the interactions can be studied using both full-length AQPs and AQP peptides corresponding to the regulatory protein binding sites. However, full-length AQPs gave better reproducibility between methods and for the first time revealed that AQP0 binds CaM in a cooperative manner, which was not seen in experiments using peptides. Our study highlights that, while peptides are great tools for locating binding sites and pinpointing interacting residues, full-length proteins may give additional insights, such as binding mechanism, allostery and cooperativity, important parameters for understanding protein-protein mediated regulation in the cellular context. Our work provides a platform for further studies of AQP regulation that may be of interest for designing drugs that target AQP complexes as well as the development of artificial bio-mimetic water channels for water-purification purposes.