Unraveling aquaporin interaction partners

Background

Insight into protein–protein interactions (PPIs) is highly desirable in order to understand the physiology of cellular events. This understanding is one of the challenges in biochemistry and molecular biology today, especially for eukaryotic membrane proteins where hurdles of production, purification and structural determination must be passed.

Scope of review

We have explored the common strategies used to find medically relevant interaction partners of aquaporins (AQPs). The most frequently used methods to detect direct contact, yeast two-hybrid interaction assay and co-precipitation, are described together with interactions specifically found for the selected targets AQP0, AQP2, AQP4 and AQP5.

Major conclusions

The vast majority of interactions involve the aquaporin C-terminus and the characteristics of the interaction partners are strikingly diverse. While the well-established methods for PPIs are robust, a novel approach like bimolecular fluorescence complementation (BiFC) is attractive for screening many conditions as well as transient interactions. The ultimate goal is structural evaluation of protein complexes in order to get mechanistic insight into how proteins communicate at a molecular level.

General significance

What we learn from the human aquaporin field in terms of method development and communication between proteins can be of major use for any integral membrane protein of eukaryotic origin. This article is part of a Special Issue entitled Aquaporins.     

Principles of membrane protein interactions with annular lipids deduced from aquaporin‐0 2D crystals

We have previously described the interactions of aquaporin‐0 (AQP0) with dimyristoyl phosphatidylcholine (DMPC) lipids. We have now determined the 2.5 Å structure of AQP0 in two‐dimensional (2D) crystals formed with Escherichia coli polar lipids (EPLs), which differ from DMPC both in headgroups and acyl chains. Comparison of the two structures shows that AQP0 does not adapt to the different length of the acyl chains in EPLs and that the distance between the phosphodiester groups in the two leaflets of the DMPC and EPL bilayers is almost identical. The EPL headgroups interact differently with AQP0 than do those of DMPC, but the acyl chains in the EPL and DMPC bilayers occupy similar positions. The interactions of annular lipids with membrane proteins seem to be driven by the propensity of the acyl chains to fill gaps in the protein surface. Interactions of the lipid headgroups may be responsible for the specific interactions found in tightly bound lipids but seem to have a negligible effect on interactions of generic annular lipids with membrane proteins.     

Cooperativity and allostery in aquaporin 0 regulation by Ca2+

Aquaporin 0 (AQP0) is essential for eye lens homeostasis as is regulation of its water permeability by Ca²⁺, which occurs through interactions with calmodulin (CaM), but the underlying molecular mechanisms are not well understood. Here, we use molecular dynamics (MD) simulations on the microsecond timescale under an osmotic gradient to explicitly model water permeation through the AQP0 channel. To identify any structural features that are specific to water permeation through AQP0, we also performed simulations of aquaporin 1 (AQP1) and a pure mixed lipid bilayer under the same conditions. The relative single-channel water osmotic permeability coefficients (p_f) calculated from all of our simulations are in reasonable agreement with experiment. Our simulations allowed us to characterize the dynamics of the key structural elements that modulate the diffusion of water single-files through the AQP0 and AQP1 pores. We find that CaM binding influences the collective dynamics of the whole AQP0 tetramer, promoting the closing of both the extracellular and intracellular gates by inducing cooperativity between neighboring subunits.     

Selectable high‐yield recombinant protein production in human cells using a GFP/YFP nanobody affinity support

Recombinant protein expression systems that produce high yields of pure proteins and multi‐protein complexes are essential to meet the needs of biologists, biochemists, and structural biologists using X‐ray crystallography and cryo‐electron microscopy. An ideal expression system for recombinant human proteins is cultured human cells where the correct translation and chaperone machinery are present. However, compared to bacterial expression systems, human cell cultures present several technical challenges to their use as an expression system. We developed a method that utilizes a YFP fusion‐tag to generate recombinant proteins using suspension‐cultured HEK293F cells. YFP is a dual‐function tag that enables direct visualization and fluorescence‐based selection of high expressing clones for and rapid purification using a high‐stringency, high‐affinity anti‐GFP/YFP nanobody support. We demonstrate the utility of this system by expressing two large human proteins, TOP2α (340 KDa dimer) and a TOP2β catalytic core (260 KDa dimer). This robustly and reproducibly yields >10 mg/L liter of cell culture using transient expression or 2.5 mg/L using stable expression.     

The Arg233Lys AQP0 mutation disturbs aquaporin0-calmodulin interaction causing polymorphic congenital cataract

Calmodulin (CaM) directly interacts with the aquaporin 0 (AQP0) C-terminus in a calcium dependent manner to regulate the water permeability of AQP0. We previously identified a missense mutation (p.R233K) in the putative CaM binding domain of AQP0 C-terminus in a congenital cataract family. This study was aimed at exploring the potential pathogenesis of this mutation causative of cataract and mainly identifying how it influenced the binding of AQP0 to CaM. Wild type and R233K mutant AQP0 with EGFP-tag were transfected separately into Hela cells to determine the expression and subcellular localizations. The co-immunoprecipitation (CoIP) assay was used to detect the interaction between AQP0 and CaM. AQP0 C-terminus peptides were synthesized with and without R233K, and the binding abilities of these peptides to CaM were assessed using a fluorescence binding assay. Localizations of wild type and R233K mutant AQP0 were determined from EGFP fluorescence, and the chimeric proteins were both localized abundantly in the plasma membrane. Protein expression levels of the culture cells showed no significant difference between them. The results from CoIP assay implied that R233K mutant presented more weakly in association with CaM than wild type AQP0. The AQP0 C-terminal mutant peptide was found to have 2.5-fold lower binding affinity to CaM than wild type peptide. These results suggested that R233K mutation did not affect the expression, location and trafficking of the protein but did influence the interaction between AQP0 and CaM. The binding affinity of AQP0 C-terminus to CaM was significantly reduced. Due to lack of the modulation of the Ca2+-calmodulin complex, the water permeability of AQP0 was subsequently augmented, which might lead to the development of this cataract.     

Noncanonical binding of calmodulin to aquaporin-0: implications for channel regulation

Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homotetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens, where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show that CaM binds the AQP0 C-terminal domain in a calcium-dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a noncanonical fashion, suggesting a form of cooperativity between AQP0 monomers. Based on these results, we derive a structural model of the AQP0/CaM complex, which suggests CaM may be inhibitory to channel permeability by capping the vestibules of two monomers within the AQP0 tetramer. Finally, phosphorylation within AQP0's CaM binding domain inhibits the AQP0/CaM interaction, suggesting a temporal regulatory mechanism for complex formation.     

Native electrophoretic techniques to identify protein–protein interactions

Permanent protein–protein interactions are commonly identified by co‐purification of two or more protein components using techniques like co‐immunoprecipitation, tandem affinity purification and native electrophoresis. Here we focus on blue‐native electrophoresis, clear‐native electrophoresis, high‐resolution clear‐native electrophoresis and associated techniques to identify stable membrane protein complexes and detergent‐labile physiological supercomplexes. Hints for dynamic protein–protein interactions can be obtained using two‐hybrid techniques but not from native electrophoresis and other protein isolation techniques except after covalent cross‐linking of interacting proteins in vivo prior to protein separation.     

Importance of the interaction protein–protein of the CaM–PDE1A and CaM–MLCK complexes in the development of new anti‐CaM drugs

Protein–protein interactions play central roles in physiological and pathological processes. The bases of the mechanisms of drug action are relevant to the discovery of new therapeutic targets. This work focuses on understanding the interactions in protein–protein–ligands complexes, using proteins calmodulin (CaM), human calcium/calmodulin‐dependent 3′,5′‐cyclic nucleotide phosphodiesterase 1A active human (PDE1A), and myosin light chain kinase (MLCK) and ligands αII–spectrin peptide (αII–spec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM (hCaM M124C–mBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaM–protein complex under analysis. For the Ca²⁺–CaM, Ca²⁺–CaM–PDE1A, and Ca²⁺–CaM–MLCK complexes, CPZ apparent dissociation constants (K_ds) were 1.11, 0.28, and 0.55 μM, respectively; and for MBC K_ds were 1.43, 1.10, and 0.61 μM, respectively. In competition experiments the addition of calmodulin binding peptide 1 (αII–spec) to Ca²⁺–hCaM M124C–mBBr quenched the fluorescence (K_d = 2.55 ± 1.75 pM) and the later addition of MBC (up to 16 μM) did not affect the fluorescent signal. Instead, the additions of αII–spec to a preformed Ca²⁺–hCaM M124C–mBBr–MBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca²⁺–CaM. In addition, docking studies were performed for all complexes with both ligands showing an excellent correlation with experimental data. These experiments may help to explain why in vivo many CaM drugs target prefer only a subset of the Ca²⁺–CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands. Copyright © 2013 John Wiley & Sons, Ltd.     

Structural Determinants of Oligomerization of the Aquaporin-4 Channel

The aquaporin (AQP) family of integral membrane protein channels mediate cellular water and solute flow. Although qualitative and quantitative differences in channel permeability, selectivity, subcellular localization, and trafficking responses have been observed for different members of the AQP family, the signature homotetrameric quaternary structure is conserved. Using a variety of biophysical techniques, we show that mutations to an intracellular loop (loop D) of human AQP4 reduce oligomerization. Non-tetrameric AQP4 mutants are unable to relocalize to the plasma membrane in response to changes in extracellular tonicity, despite equivalent constitutive surface expression levels and water permeability to wild-type AQP4. A network of AQP4 loop D hydrogen bonding interactions, identified using molecular dynamics simulations and based on a comparative mutagenic analysis of AQPs 1, 3, and 4, suggest that loop D interactions may provide a general structural framework for tetrameric assembly within the AQP family.     

The third dimension for protein interactions and complexes

Interaction discovery methods, such as the two-hybrid system and affinity purification, suggest thousands of protein–protein interactions. Structural biology provides atomic details for many interactions but, to date, there has been limited discussion of how these two fields complement each other. Here, we apply a structural perspective to interpret interactions discovered by different techniques. This perspective reveals indirect interactions in two-hybrid systems, instances where molecular labels might obstruct interfaces, and possible explanations for why certain promiscuous proteins interact with many others. It also highlights that some methods favour tight complexes whereas others favour interactions of a more transient nature. We conclude by discussing how a combination of interaction discovery and structural biology will enhance our understanding of complex cellular processes.     

Simultaneous visualization of two protein complexes in a single plant cell using multicolor fluorescence complementation analysis

Bimolecular fluorescence complementation (BiFC) is an approach used to analyze protein–protein interaction in vivo, in which non-fluorescent N-terminal and C-terminal fragments of a fluorescent protein are reconstituted to emit fluorescence only when they are brought together by interaction of two proteins to fuse both fragments. A method for simultaneous visualization of two protein complexes by multicolor BiFC with fragments from green fluorescent protein (GFP) and its variants such as cyan and yellow fluorescent proteins (CFP and YFP) was recently reported in animal cells. In this paper we describe a new strategy for simultaneous visualization of two protein complexes in plant cells using the multicolor BiFC with fragments from CFP, GFP, YFP and a red fluorescent protein variant (DsRed-Monomer). We identified nine different BiFC complexes using fragments of CFP, GFP and YFP, and one BiFC complex using fragments of DsRed-Monomer. Fluorescence complementation did not occur by combinations between fragments of GFP variants and DsRed-Monomer. Based on these findings, we achieved simultaneous visualization of two protein complexes in a single plant cell using two colored fluorescent complementation pairs (cyan/red, green/red or yellow/red).     

Distribution and Dynamics of Gap Junction Channels Revealed in Living Cells

To study the structural composition and dynamics of gap junctions in living cells, we tagged their subunit proteins, termed connexins, with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. Tagged connexins assembled normally and channels were functional. High-resolution fluorescence images of gap junction plaques assembled from CFP and YFP tagged connexins revealed that the mode of channel distribution is strictly dependent on the connexin isoforms. Co-distribution as well as segregation into well-separated domains was observed. Based on accompanying studies we propose that channel distribution is regulated by intrinsic, connexin isoform specific signals. High-resolution time-lapse images revealed that gap junctions, contrary to previous expectations, are dynamic assemblies of channels. Channels within clusters and clusters themselves are mobile and constantly undergo structural rearrangements. Movements are complex and allow channels to move, comparable to other plasma membrane proteins not anchored to cytoskeletal elements. Comprehensive analysis, however, demonstrated that gap junction channel movements are not driven by diffusion described to propel plasma membrane protein movement. Instead, recent studies suggest that movements of gap junction channels are indirect and predominantly propelled by plasma membrane lipid flow that results from metabolic endo- and exocytosis.     

Distribution and Dynamics of Gap Junction Channels Revealed in Living Cells

To study the structural composition and dynamics of gap junctions in living cells, we tagged their subunit proteins, termed connexins, with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. Tagged connexins assembled normally and channels were functional. High-resolution fluorescence images of gap junction plaques assembled from CFP and YFP tagged connexins revealed that the mode of channel distribution is strictly dependent on the connexin isoforms. Co-distribution as well as segregation into well-separated domains was observed. Based on accompanying studies we propose that channel distribution is regulated by intrinsic, connexin isoform specific signals. High-resolution time-lapse images revealed that gap junctions, contrary to previous expectations, are dynamic assemblies of channels. Channels within clusters and clusters themselves are mobile and constantly undergo structural rearrangements. Movements are complex and allow channels to move, comparable to other plasma membrane proteins not anchored to cytoskeletal elements. Comprehensive analysis, however, demonstrated that gap junction channel movements are not driven by diffusion described to propel plasma membrane protein movement. Instead, recent studies suggest that movements of gap junction channels are indirect and predominantly propelled by plasma membrane lipid flow that results from metabolic endo- and exocytosis.     

DockTrina: Docking triangular protein trimers

In spite of the abundance of oligomeric proteins within a cell, the structural characterization of protein–protein interactions is still a challenging task. In particular, many of these interactions involve heteromeric complexes, which are relatively difficult to determine experimentally. Hence there is growing interest in using computational techniques to model such complexes. However, assembling large heteromeric complexes computationally is a highly combinatorial problem. Nonetheless the problem can be simplified greatly by considering interactions between protein trimers. After dimers and monomers, triangular trimers (i.e. trimers with pair‐wise contacts between all three pairs of proteins) are the most frequently observed quaternary structural motifs according to the three‐dimensional (3D) complex database. This article presents DockTrina, a novel protein docking method for modeling the 3D structures of nonsymmetrical triangular trimers. The method takes as input pair‐wise contact predictions from a rigid body docking program. It then scans and scores all possible combinations of pairs of monomers using a very fast root mean square deviation test. Finally, it ranks the predictions using a scoring function which combines triples of pair‐wise contact terms and a geometric clash penalty term. The overall approach takes less than 2 min per complex on a modern desktop computer. The method is tested and validated using a benchmark set of 220 bound and seven unbound protein trimer structures. DockTrina will be made available at http://nano‐d.inrialpes.fr/software/docktrina . Proteins 2014; 82:34–44. © 2013 Wiley Periodicals, Inc.     

cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera

To study the membrane mobility of aquaporin water channels, clones of stably transfected LLC-PK1 cells were isolated with plasma membrane expression of GFP-AQP1 and GFP-AQP2, in which the green fluorescent protein (GFP) was fused upstream and in-frame to each aquaporin (AQP). The GFP fusion did not affect AQP tetrameric association or water transport function. GFP-AQP lateral mobility was measured by irreversibly bleaching a spot (diameter 0.8 microm) on the membrane with an Argon laser beam (488 nm) and following the fluorescence recovery into the bleached area resulting from GFP translational diffusion. In cells expressing GFP-AQP1, fluorescence recovered to >96% of its initial level with t(1/2) of 38 +/- 2 s (23 degrees C) and 21 +/- 1 s (37 degrees C), giving diffusion coefficients (D) of 5.3 and 9.3 x 10(-11) cm(2)/s. GFP-AQP1 diffusion was abolished by paraformaldehyde fixation, slowed >50-fold by the cholesterol-binding agent filipin, but not affected by cAMP agonists. In cells expressing GFP-AQP2, fluorescence recovered to >98% with D of 5.7 and 9.0 x 10(-11) cm(2)/s at 23 degrees C and 37 degrees C. In contrast to results for GFP-AQP1, the cAMP agonist forskolin slowed GFP-AQP2 mobility by up to tenfold. The cAMP slowing was blocked by actin filament disruption with cytochalasin D, by K(+)-depletion in combination with hypotonic shock, and by mutation of the protein kinase A phosphorylation consensus site (S256A) at the AQP2 C-terminus. These results indicate unregulated diffusion of AQP1 in membranes, but regulated AQP2 diffusion that was dependent on phosphorylation at serine 256, and an intact actin cytoskeleton and clathrin coated pit. The cAMP-induced immobilization of phosphorylated AQP2 provides evidence for AQP2-protein interactions that may be important for retention of AQP2 in specialized membrane domains for efficient membrane recycling.     

Fluorescence labeling, purification, and immobilization of a double cysteine mutant calmodulin fusion protein for single-molecule experiments

We present a method of labeling and immobilizing a low-molecular-weight protein, calmodulin (CaM), by fusion to a larger protein, maltose binding protein (MBP), for single-molecule fluorescence experiments. Immobilization in an agarose gel matrix eliminates potential interactions of the protein and the fluorophore(s) with a glass surface and allows prolonged monitoring of protein dynamics. The small size of CaM hinders its immobilization in low-weight-percentage agarose gels; however, fusion of CaM to MBP via a flexible linker provides sufficient restriction of translational mobility in 1% agarose gels. Cysteine residues were engineered into MBP.CaM (MBP-T34C,T110C-CaM) and labeled with donor and acceptor fluorescent probes yielding a construct (MBP.CaM-DA) which can be used for single-molecule single-pair fluorescence resonance energy transfer (spFRET) experiments. Mass spectrometry was used to verify the mass of MBP.CaM-DA. Assays measuring the activity of CaM reveal minimal activity differences between wild-type CaM and MBP.CaM-DA. Single-molecule fluorescence images of the donor and acceptor dyes were fit to a two-dimensional Gaussian function to demonstrate colocalization of donor and acceptor dyes. FRET is demonstrated both in bulk fluorescence spectra and in fluorescence trajectories of single MBP.CaM-DA molecules. The extension of this method to other biomolecules is also proposed.     

Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signaling using spectral variants of the green fluorescent protein

BACKGROUND: Fluorescence resonance energy transfer (FRET) is a powerful technique for measuring molecular interactions at Angstrom distances. We present a new method for FRET that utilizes the unique spectral properties of variants of the green fluorescent protein (GFP) for large-scale analysis by flow cytometry. METHODS: The proteins of interest are fused in frame separately to the cyan fluorescent protein (CFP) or the yellow fluorescent protein (YFP). FRET between these differentially tagged fusion proteins is analyzed using a dual-laser FACSVantage cytometer. RESULTS: We show that homotypic interactions between individual receptor chains of tumor necrosis factor receptor (TNFR) family members can be detected as FRET from CFP-tagged receptor chains to YFP-tagged receptor chains. Noncovalent molecular complexation can be detected as FRET between fusions of CFP and YFP to either the intracellular or extracellular regions of the receptor chains. The specificity of the assay is demonstrated by the absence of FRET between heterologous receptor pairs that do not biochemically associate with each other. Interaction between a TNFR-like receptor (Fas/CD95/Apo-1) and a downstream cytoplasmic signaling component (FADD) can also be demonstrated by flow cytometric FRET analysis. CONCLUSIONS: The utility of spectral variants of GFP in flow cytometric FRET analysis of membrane receptors is demonstrated. This method of analyzing FRET allows probing of noncovalent molecular interactions that involve both the intracellular and extracellular regions of membrane proteins as well as proteins within the cells. Unlike biochemical methods, FRET allows the quantitative determination of noncovalent molecular associations at Angstrom level in living cells. Moreover, flow cytometry allows quantitative analyses to be carried out on a cell-by-cell basis on large number of cells. Published 2001 Wiley-Liss, Inc.     

A general strategy to characterize calmodulin-calcium complexes involved in CaM-target recognition: DAPK and EGFR calmodulin binding domains interact with different calmodulin-calcium complexes

Calmodulin (CaM) is a ubiquitous Ca²⁺ sensor regulating many biochemical processes in eukaryotic cells. Its interaction with a great variety of different target proteins has led to the fundamental question of its mechanism of action. CaM exhibits four “EF hand” type Ca²⁺ binding sites. One way to explain CaM functioning is to consider that the protein interacts differently with its target proteins depending on the number of Ca²⁺ ions bound to it. To test this hypothesis, the binding properties of three entities known to interact with CaM (a fluorescent probe and two peptide analogs to the CaM binding sites of death associated protein kinase (DAPK) and of EGFR) were investigated using a quantitative approach based on fluorescence polarization (FP). Probe and peptide interactions with CaM were studied using a titration matrix in which both CaM and calcium concentrations were varied. Experiments were performed with SynCaM, a hybrid CaM able to activate CaM dependent enzymes from mammalian and plant cells. Results show that the interaction between CaM and its targets is regulated by the number of calcium ions bound to the protein, namely one for the DAPK peptide, two for the probe and four for the EGFR peptide. The approach used provides a new tool to elaborate a typology of CaM-targets, based on their recognition by the various CaM-Ca_n (n = 0-4) complexes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Rapid expression screening of eukaryotic membrane proteins in Pichia pastoris

The overexpression of milligram quantities of protein remains a key bottleneck in membrane protein structural biology. A challenge of particular difficulty has been the overproduction of eukaryotic membrane proteins. In order to cope with the frequently poor expression levels associated with these challenging proteins, it is often necessary to screen a large number of homologues to find a well expressing clone. To facilitate this process using the heterologous, eukaryotic expression host Pichia pastoris, we have developed a simple fluorescent induction plate‐screening assay that allows for the rapid detection of well expressing clones of eukaryotic membrane proteins that have been fused to GFP. Using a eukaryotic membrane protein known to express well in P. pastoris (human aquaporin 4) and homologues of the ER associated membrane protein phosphatidylethanolamine N‐methyltransferase (PEMT), we demonstrate that when a large number of clones are screened, a small number of highly expressing “jackpot” clones can be isolated. A jackpot PEMT clone resulted in 5 mg/L yield after purification. The method allows for the facile simultaneous screening of hundreds of clones providing an alternate to in‐culture screening and will greatly accelerate the search for overexpressing eukaryotic membrane proteins.