Protein–protein interactions in AQP regulation – biophysical characterization of AQP0–CaM and AQP2–LIP5 complex formation

Abstract

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, fulllength 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.

A microfluidic strategy for the detection of membrane protein interactions

“…This implies strong positive cooperativity of CaM binding to AQP0 in line with previous findings. 28 In the absence of strong positive cooperativity, one CaM per AQP0 tetramer would dominate if AQP0 is in excess relative to CaM (Fig. 4b).…”

“…The interaction between CaM and AQP0 has been studied for a range of organisms and sometimes between homologs from different species. [7][8][9]22,28,37 Interestingly, a wide range of K d values have been reported for the interaction from below 100 nM to 40 μM, with most values in the low micromolar range, 8,22,37 with micromolar affinities and strong positive cooperativity measured for the interaction between CaM and full-length AQP0 by thermophoresis. 28 We have determined the K d for the interaction through three direct measurements of the physical properties of the interaction partners (the electrophoretic mobilities of CaM and AQP0, and the diffusion coefficient of CaM), these all report on a K d of 0.3-1.3 μM.…”

“…In most reports of CaM-ligand interactions, CaM interacts with a single copy of the target sequence. 8 Depending on the technique, CaM has been reported to interact with one or two C-termini of the AQP0 subunits, 8,22,28,37 and it has been suggested that the specifics of the CaM-AQP0 interaction vary between species. 9 In addition to the use of truncated versus full length AQP0, the spread in the reported K d values for the interaction may further arise from differences solution conditions used for each study, including composition, pH, ionic strength, and protein concentration, NMR for instance employs CaM concentrations in the hundreds of μM.…”

“…43 AQP0 and AQP2 were purified as described previously. 28 Human CaM was expressed in Escherichia coli and purified as previously described. 26 As human CaM does not contain any native cysteines, Ser 17 of CaM was replaced by cysteine, which allowed for labelling with cysteine-reactive dyes.…”

“…7,8,22,27 Recent methodological developments such as microscale thermophoresis have enabled studies of the interaction between full-length AQP0 and CaM, providing novel insights into the interaction mode. 28 Nevertheless, our understanding of the biophysical properties of the AQP0-CaM complex is still limited, due to the lack of strategies to determine key physicochemical parameters such as the charge and size of such integral membrane protein complexes. Determination of the equilibrium parameters for CaM binding to full-length AQP0 and characterisation of the physicochemical properties defining the complex are therefore important for a full understanding of how CaM interacts with AQP0.…”