Combined Inhibitory Effects of Citrinin, Ochratoxin-A, and T-2 Toxin on

Article Cite This: J. Phys. Chem. B 2019, 123, 5755−5768

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Combined Inhibitory Effects of Citrinin, Ochratoxin-A, and T‑2 Toxin on Aquaporin‑2 Nikhil Maroli,*,† Achuth Jayakrishnan,‡ Renuka Ramalingam Manoharan,‡ Ponmalai Kolandaivel,*,§ and Kadirvelu Krishna‡ Computational Biology Division and ‡Molecular Immunology and Toxicology Division, DRDO BU CLS, Coimbatore 641046, Tamil Nadu, India § Periyar University, Salem 636011, Tamil Nadu, India Downloaded via KEAN UNIV on July 22, 2019 at 08:08:47 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

S Supporting Information *

ABSTRACT: Aquaporins form a large family of transmembrane protein channel that facilitates selective and fast water transport across the cell membrane. The inhibition of aquaporin channels leads to many water-related diseases such as nephrogenic diabetes insipidus, edema, cardiac arrest, and stroke. Herein, we report the molecular mechanism of mycotoxins (citrinin, ochratoxin-A, and T-2 mycotoxin) inhibition of aquaporin-2 (AQP2) and arginine vasopressin receptor 2. Molecular docking, molecular dynamics simulations, quantum chemical calculations, residue conservationcoupling analysis, sequence alignment, and in vivo studies were utilized to explore the binding interactions between the mycotoxins and aquaporin-2. Theoretical studies revealed that the electrostatic interactions induced by the toxins pulled the key residues (187Arg, 48Phe, 172His, and 181Cys) inward, hence reduced the pore diameter and water permeation. The permeability coefficient of the channel was reduced from native ((3.32 ± 0.75) × 10−14 cm3/s) to toxin-treated AQP2 ((1.08 ± 0.03) × 10−14 cm3/s). The hydrogen bonds interruption and formation of more hydrogen bonds with toxins also led to the reduced number of water permeation. Further, in vivo studies showed renal damages and altered level of aquaporin expression in mycotoxin-treated Mus musculus. Furthermore, the multiple sequence alignments among the model organism along with evolutionary coupling analysis provided the information about the interdependences of the residues in the channel.

1. INTRODUCTION Aquaporins are transmembrane protein channel that maintains water homeostasis in human as well as in animals. They form a tetramer assembly in the cell membrane and facilitate water flow along osmotic gradients. 1 Aquaporins are widely distributed in bacteria, plants, and animals, and 13 aquaporins have been identified in mammalian cells.2,3 Aquaporins are classified into three groups based on its selectivity and permeability: classical aquaporins, aquaglyceroporins, and superaquaporins. The classical aquaporins (AQPs 0, 1, 2, 4, 5, 6, and 8) have exclusive selectivity for water, whereas aquaglyceroporins (AQPs 3, 7, 9, and 10) are permeable for water, glycerol, and other small molecules. The superaquaporins (AQP11 and 12) have high selectivity toward water, although comprises less homology sequence similarity with other aquaporins.3 Aquaporins mediate osmotic water transport across the plasma membrane, neuroexcitation, and cell migration at the cellular level. Also, aquaporin controls the glycerol transport-regulated cell proliferation, epidermal water retention, and adipocyte metabolism.4−6 The extracellular and cytoplasmic vestibules of each aquaporin monomers are linked through a central amphipathic pore region. The signature motifs of the aquaporin are sequence of amino acids called © 2019 American Chemical Society

NPA (Asn-Pro-Ala) motifs. NPA motifs found at the halflength of each monomer unit and its carboxy terminal half acts as hydrogen donor and acceptor, respectively, that control water transportation.7−9 The extracellular vestibule region contains a narrow region called the ar/R region, which is made up of aromatic arginine residues that prevent proton conduction through the pore. The AQP2 monomer units are composed of six transmembrane helices and two short loops. The half-helices fold into the channel and are surrounded by the six long transmembrane helices and expressed in cells as a tetramer.9−12 Water reabsorption in the kidney collecting duct is a crucial event for maintaining the body homeostasis. AQP2 expressed in the apical membrane of the collecting duct by regulation of vasopressin, which is secreted from the pituitary gland.13 arginine vasopressin receptor 2 (AVPR2) is a receptor protein that belongs to the subfamily of G-protein-coupled receptors. AVPR2 is expressed in the kidney, predominantly in the membrane of cells of distal convoluted tubules and collecting Received: April 24, 2019 Revised: June 15, 2019 Published: June 16, 2019 5755

DOI: 10.1021/acs.jpcb.9b03829 J. Phys. Chem. B 2019, 123, 5755−5768

Article

The Journal of Physical Chemistry B

mycotoxins on aquaporin channel due to its well-established characteristics like cell membrane penetration, which includes the blood−brain barrier. In this regard, we have subjected the toxins, based on the IC50 value, to a group of healthy albino mice of same age, sex, and average weight to investigate the AQP2 and AVPR2 expression and to understand the histological changes induced by these mycotoxins. Further, molecular dynamics (MD) simulation of AQP2 protein in POPE lipid bilayers was carried out to estimate the biological parameters such as osmotic and diffusive permeability constants. The interaction and mechanisms involved in the inhibition of the AQP2 by mycotoxins were analyzed using MD simulations, MM-PBSA, and DFT calculations. Furthermore, residue conservation analysis was used to derive a unique binding location of the aquaporin for future therapeutic applications.

ducts. An increase of vasopressin in the blood leads to higher water permeability in the collecting duct principal cells by stimulating AVPR2; the detailed mechanism of AVPR2 and AQP2 trafficking is described elsewhere.14,15 Activation of Gcoupled proteins results in translocation of AQP2 from intracellular storage vesicles to the apical membrane, leading to high water permeability in the renal cells. The withdrawal of vasopressin from AVPR2 leads to the impermeable water state.14−19 The AQP2 dysregulation is commonly associated with clinical conditions that exhibit body−water balance disturbance, electrolyte’s disturbance, nephrotic syndrome, hereditary nephrogenic diabetes insipidus (NDI), acute and chronic renal failure, lithium-induced NDI, hepatic cirrhosis, congestive heart failure, and ureteral obstruction.20−24 The mutations in AQP2 cause NDI that is characterized by the inability of patients to concentrate urine despite normal antidiuretic hormone concentrations in the blood. Similarly, AQP2 inhibition by toxins and heavy metals also leads to the same outcome. AQP2 is also involved in acquired NDI due to the application of lithium, which is widely used in bipolar disorder treatment.25 Lithium enters the cells that express AQP2 via the epithelial sodium channel in the apical membrane and accumulates intracellularly and inhibit the expression as well as function of the channel.26,27 The mice models with deleted AVPR2 genes showed characteristic symptoms of NDI such as urinary concentrating defects and dilatation of the renal pelvis.28 Presently, there is no cure for NDI apart from the restricted salt diet combined with administration of hydrochlorothiazide diuretics to reduce urine output.25 Mycotoxin-induced organ toxicity also leads to NDI and associated kidney diseases; the occurrence of mycotoxins toxicity is very high due to its presence in food materials. Mycotoxins are toxic fungal secondary metabolites capable of causing diseases that eventually lead to death. Mycotoxins are nonvolatile and resistant to degradation in normal environmental conditions such as light and temperature, but it is degradable in strong acid or alkaline conditions.29,30 The metabolism of mycotoxins occurs mainly in the liver, and both liver and nonliver metabolisms produce various toxic effects in animals and human. Several compounds with antioxidant properties are widely used to treat these, such as vitamins, provitamins, carotenoids, chlorophyll and its derivatives, phenolics, selenium, and synthetic compounds including butylated hydroxyanisole and butylated hydroxytoluene.29 Exposure to mycotoxins leads to severe diseases such as stachybotryotoxicosis and alimentary toxic aleukia.31−33 T-2 toxin belongs to the trichothecenes mycotoxins that are often encountered in natural contaminants. T-2 toxin interferes in the T-cell-mediated functions and induces type IV hypersensitivity in animals. Ochratoxin A (OTA) and citrinin (CTN) are ubiquitous mycotoxins produced by fungi, and they are the nephrotoxic and primary etiological agents responsible for human balkan endemic nephropathy (BEN) and etiology of endemic nephropathy.34−36 The effect of these mycotoxins and the mechanisms of action on the renal system, especially on water channels, are not yet understood in the scientific world. The widespread distribution of aquaporin provides a great opportunity as a therapeutic target. However, these are not easily accessible for small therapeutic molecules or drugs due to their unique expression and positioning in the lipid bilayer. Considering this, we have assessed the effect of interaction of

2. MATERIALS AND METHODS 2.1. Protein−Membrane System Preparation. The Xray crystal structure of aquaporin-2 tetramer was retrieved from PDB databank with PDB ID: 4NEF37 and embedded in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) lipid bilayer. The missing residues were added to the AQP2 prior to the lipid bilayer insertions using GalaxyFill.38 The protein was aligned to the membrane bilayer using the OPM web server, and POPE lipid bilayers with 100 Å length in X and Y directions were obtained from CHARMM-GUI server.39−41 A water box of thickness 20 Å was added below and above the protein−membrane complex, and the system was neutralized with KCl ions; the entire system consists of 99 868 atoms. The tetramer structures associated with toxin molecules were generated with YASARA Structure software,42 and the same procedure was adapted to preparing the AQP2toxin-bound form. 2.2. Molecular Dynamics Simulation. NAMD 2.1143 was used to perform equilibrium molecular dynamics simulation with CHARMM36 forcefield44,45 and TIP3P water model. The parameters for the toxin molecules were obtained from cgenparamchem,46,47 and toxin molecules were optimized at the B3LYP/6-31G(d,p) level of theory48−50 using Gaussian 09 software package.51 A cutoff distance of 12 Å with a switching distance of 10 Å was employed for van der Waals interaction by applying the particle mesh Ewald (PME) method.52 MD simulation of the systems was carried out in three phases. In the first phase, water, ions, protein, and lipid headgroups were fixed and the lipid tail was minimized and equilibrated for 1 ns to achieve melting lipid tail. NPT simulation (100 ns) with a time step of 2 fs was performed after NVT and NPT equilibrations, maintaining a temperature of 310 K and a pressure of 1 bar using the Langevin piston algorithm53 method. A 100 ns independent MD simulation of ligand molecules with AQP2 at the pore region and a 200 ns simulation of AQP2 with ligands in the midregion of the monomer unit were performed using Gromacs 2016.1.54,55 Minimization of the protein−ligand system was achieved by the steepest descent method followed by an NVT and NPT ensemble equilibration. The production run (NPT) and the trajectory were saved every 100 ps. The criteria for hydrogen bond analysis were assigned based on the previous reports.56−63 Water permeability through biological channels was computed using the continuous-time random walk model (CTRW).64−66 The permeability constant pd was calculated 5756


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where EA(AB) and EB(AB) represent the energies of individuals A and B, respectively, in the compound geometries, and EA**(AB) and EB**(AB) represent the energies of A and B in the respective ghost orbitals. All of the above calculations were performed using Gaussian 09 program package. 2.5. In Vivo Toxicity Analysis of Mycotoxins. Male BALB/c mice (8−12 weeks old) weighing 15−25 g were used in the present study. For a week, the animals were placed in an individual metabolic cage for acclimation and they were divided into four groups with three mice per group for the study. The first group was subjected to physiological endotoxin-free saline intravenously and served as the control group. The remaining three groups were immunized intravenously with mycotoxins, namely, citrinin, T-2 toxin, and ochratoxin-A, as 8 μg/kg body weight in phosphate-buffered saline (PBS) (w/v), respectively. The animals were monitored for 24 h and sacrificed for the study. All of the experiments were carried out in strict accordance with the recommendation based on the Institutional Animal Ethics Committee (IAEC) guidelines. 2.6. Tissue Preparation and Histopathology. The mice were sacrificed and the kidneys were clamped and removed for preparation of crude membrane fractions. The dissected organ was washed with saline and fixed by immersion in 4% paraformaldehyde (w/v) in 0.1 M sodium phosphate buffer (pH 7.3). The tissues were briefly cleared in dimethylsulfoxide followed by PBS wash (pH 7.2) and cryoprotected through 30% sucrose infiltration (w/v in PBS). The tissues were embedded in OCT compound, and frozen sections were cut at −20 °C with cryostat microtome (MICROM). Kidney tissues were entrenched in paraffin blocks for sectioning into 4 μm sections, stained with hematoxylin and eosin staining, and visualized under a fluorescent microscope (Life Technologies). 2.7. Immunoblotting Analysis. All tissues were minced finely and homogenized in 1 mL of dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, and containing the following protease inhibitors: 8.5 mM leupeptin, 1 mM phenylmethylsulfonyl fluoride). The homogenate was centrifuged at 4000g for 15 min at 4 °C, and the steps were repeated thrice with successive pellet fractions. The supernatants were pooled and centrifuged at 17 500g for 30 min. The resultant pellet was resuspended in approximately 1 mL of dissecting buffer and protein concentration was estimated. The samples were then diluted with Laemmli sample buffer (2% sodium dodecyl sulfate (SDS), 30% glycerol, and 1 M tris(hydroxymethyl) aminomethane, pH 6.8, 0.0002 g bromophenol blue) to a final concentration of 1 mg/mL. These membrane fractions were loaded at 30 μg/lane onto 12% SDS/PAGE gels and electrophoretically separated (Bio-Rad, Mini-PROTEAN). The proteins were then transferred onto a nitrocellulose membrane by electroelution, and the unoccupied sites were blocked with 5% dried skimmed milk (w/v in PBS) at 4 °C overnight. The membrane was washed successively with PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20, pH 7.5) and PBS. The membranes were then incubated separately with AVPR2 (E-AB-36457, ELAB Sciences) and AQP2 (E-AB-30540, ELAB Sciences) antibodies at 1:2000 dilution (w/v in PBS) for 1 h at 37 °C. The membranes were washed as mentioned above and incubated with antirabbit horseradish peroxidaseconjugated secondary antibody (1:2000 dilution) for 1 h at 37 °C. Followed by stringent washing, the membrane was

from the equilibrium molecular dynamics simulation as follows: pd = Vw ×k 0

(1)

k 0 = N±/(2 × nm × tsim)

(2)

where k0 is the number of water molecules that cross the channel per unit time, Vw is the average volume of a single water molecule, nm represents the number of monomer, and tsim is the duration of the simulation. Subsequently, the osmotic permeability coefficient (pf) was derived from collective diffusion constant. Dn = ⟨n2(t )⟩/2t

(3)

where ⟨n(t)2⟩ is the mean-square displacement (MSD) of n(t), and the osmotic permeability constant (pf) was calculated as

pf = Vw ×Dn

(4)

The damping coefficient in the Langevin thermostat was set to 1 p s−1 for all of the simulations. 2.3. MMPBSA. The binding energy and individual residue contribution toward the binding energy were calculated using the molecular mechanics Poisson−Boltzmann surface area (MMPBSA) implemented in g_mmpbsa.67,68 Binding free energy of protein−ligand complex in the solvent is expressed as ΔG binding = Gcomplex − (Gprotein + G ligand)

(5)

where Gcomplex is the total free energy of protein−ligand complex, and Gprotein and Gligand are the total energies of separated protein and ligand in the solvent, respectively. ΔG binding = ⟨ΔEMM⟩ + ⟨ΔGsolv ⟩ − ⟨T ΔS⟩

(6)

⟨ΔGsolv ⟩ = ⟨ΔGpb⟩ + ⟨ΔGnp⟩

(7)

⟨ΔEMM⟩ = ⟨ΔEvdw ⟩ + ⟨ΔEele⟩

(8)

where ⟨ΔEMM⟩ is the total molecular mechanic’s energy in the gas phase, ⟨ΔGsolv⟩ is salvation free energy, TΔS is entropy, and ⟨ΔEMM⟩ is the sum of electrostatic and van der Waals interaction energies. Polar contributions were calculated using the PB model, and nonpolar energy is estimated by solvent accessible surface area (SASA). TΔS is considered as negligible. 2.4. Quantum Chemical Calculation. The density functional theory method was applied to study the interaction of individual active sites of the mycotoxins. Becke’s threeparameter (B3) exchange functional along with Lee−Yang− Parr’s (LYP) gradient-corrected correlation functional represented as B3LYP of the density functional theory method with 6-31G (d,p) basis set has been used to optimize all of the active site regions with toxins. Interaction energy calculations have been performed to study the binding nature of residues using the same level of theory. The basis set superposition error method was used to correct the interaction energies along with counterpoise69,70 correction method of Boys and Bernardi, as shown below ΔE = EAB − (EA + E B) + CP

CP = (EA (AB) − EA **(AB)) + ((E B(AB) − E B**(AB)) 5757


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Figure 1. Docked binding sites of (a) citrinin, (b) ochratoxin-A, and (c) T-2 mycotoxins. The hydrogen bond and hydrophobic interactions at the binding site for (d) citrinin, (e) ochratoxin-A, and (f) T-2 mycotoxins.

Figure 2. Aquaporin exhibit in tetramer form (a) and each monomer unit (b) transports water across the membrane. To simulate the real-time biological system, we have embedded the AQP2 in lipid membrane and added water at the top and bottom. The colored ribbon in (c) represents the AQP2, water molecules represent a cornflower blue ball, and POPE lipid bilayer is shown as a light brown stick model. The initial conformation was used to study the transportation and inhibition mechanism of the channel with mycotoxins. (d) Superposed ribbon structure of AQP2 monomer unit; green represents the native conformation, and orange represents the toxin-treated AQP2. The NPA motifs are represented as stick models, and minor transformation from a native structure are observed.

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Figure 3. (a−d) Positioning of the residues Arg187, Phe48, His172, and Cys181. (b) The distance between the residues was reduced after interacting with toxin. (d) Marked residues from the pore region.

developed with OPD dissolved in Tris-Cl (pH 7.2). β-Actin served as a reference protein in all of the experiments. 2.8. Expression of Aquaporin-2 Messenger RNA (mRNA). The kidney tissue sample was prepared with the aid of Trizol (Sigma-Aldrich), and total RNA was isolated according to the manufacturer’s instructions. Nearly 4 μg of RNA (both treated and control) was employed for complementary DNA (cDNA) synthesis with first-strand cDNA Synthesis Kit (GE27-9261-01, GE) using oligo dT primers. cDNA synthesis was performed under the following conditions: 25 °C for 10 min, 42 °C for 60 min, followed by a final inactivation step at 95 °C for 5 min. cDNA (1 μL) was employed as template, and amplification was carried out using microAmp 96-well reaction plate using SYBR Premix Ex Taq II (TAKARA, Japan). Real-time analysis of aquaporin-2 and AVPR2 mRNA expression was carried out in Roche’s light cycler 480 RT-PCR System. The amplification conditions were 35 cycles at 94 °C for 20 s, 51 °C for 20 s, and 68 °C for 30 s. The primers were derived from sequences with GenBank accession no. NM_009699.3; in-house-designed primers for AQP2 (GR-F/R) as well as AVRP2 with gene runner and GAPDH-specific primers were used as the internal controls. The lengths of the amplification products are as follows: GRF/R (595 bp), AVPR2 (150 bp), and GAPDH (150 bp). Target mRNA was normalized to the GAPDH mRNA as an internal control in each sample, and the results were expressed as the relative ratio to the normal group average.

was analyzed by deriving the permeability coefficients, and the toxicity of the mycotoxins was evaluated using in vivo experiments conducted in albino mice. 3.1. Mycotoxins Inhibition of Aquaporin -2. 3.1.1. Molecular Docking Analysis: Binding of Mycotoxins and Interactions. Automatic docking reveals that all binding poses were within the pore region of the channel (Figure 1a−c). Citrinin was surrounded by residues such as His177, Ser122, Ile176, Gly80, Ile44, Arg187, Ala117, and Asp115. Further, strong hydrophobic interaction was seen at the binding pocket with residues Gly180, Ser122, His177, Ile44, Asp115,ala31, and Ala117. The hydrophobic and nonbonded interactions at the binding pocket generate a strong electrostatic network over the pore region. The OTA with longer chain accesses more residues than citrinin and form hydrogen bonds with Pro39 and Arg185. The interaction with the aromatic arginine residue at the pore region leads to malfunctioning of the channel. OTA was surrounded by residues such as Ile44 His177, Pro39, Ser122, Thr179, Asp115, Ala120, Val118, Ala117, Gly180, Ala31, and Ile176. Similarly, T-2 toxins create strong interactions with Val41, His177, Asn123, Ala120, Val118, Asn33, Asp115, A and Ser122. The pore region of the aquaporin constitutes Arg218, Cyc181, Phe48, and His172; these residues face each other and form the narrow part of the channel. 3.1.2. Molecular Dynamics Simulations: Dynamics of AQP2 and Inhibition Mechanism. Aquaporin-2 consists of four monomer units composed of six transmembrane helices and two short loops that form half-helices and fold into the channel (Figure 2a,b). There are NPA motifs in the loops that are formed by amino acids, asparagine (N), proline (P), and alanine (A), which stabilize the helix loops through hydrogen bonds. Due to the structural and conformational uniqueness of AQP2, we first assessed the functional changes upon interaction with mycotoxins through evaluating the changes in the secondary structure (Figure S1). The secondary structure of AQP2 monomer consists of 66.5% helix, 11.7% turn, and 21.8% coil. Citrinin and OTA-bound proteins

3. RESULTS AND DISCUSSION The binding and inhibition of mycotoxins such as citrinin, ochratoxin-A, and T-2 toxin have been evaluated using molecular docking and molecular dynamics simulations. The initial conformations were obtained from docking results and simulation provided the dynamical information about the AQP2 and AVPR2. AVPR2 is a significant receptor protein that mediates the expression of AQP2 in the kidney collecting duct. Inhibition of AVPR2 leads to the failure of AQP2 expression and functioning. Further, water dynamics of AQP2 5759


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Figure 4. (a) RMSD of backbone atoms of AQP2 and toxin-bound AQP2. The apo form of AQP2 shows comparatively fewer fluctuations than mycotoxin bound. (b) RMSD-backbone atoms of AQP2 heteromer with three toxins. Toxin-bound protein shows higher RMSD and converged at 2.1 nm, whereas native AQP2 is stable and converged at 1.4 nm. (c, d) Hydrogen bonds formed between the mycotoxins-AQP2 and -AVPR2. A larger number of hydrogen bonds are observed in all of the cases, and more details are given in Section 3.1.2.

Figure 5. Coulombic and van der Waals interaction energies between the toxin molecules and AQP2. The mycotoxins possess high nonbonded interaction energy, with AQP2 indicating its strong binding affinity.

exhibited a transition of 69.0% helix, 3.3% turn, 25.9% coil, and 1.7% 3−10 helix. The transformations from the coil to helix and turn to helix are associated with the toxin interaction within the pore region. T-2-bound monomer units showed minor changes from turn to coil as 64.4% helix, 6.7% turn, and 28.9% coil. In addition, the root-mean-square fluctuations of Cα atoms along the first two eigenvectors were calculated to understand the collective motion of the protein by principal component analysis. PCA based on Cα atoms in the protein

denoted by the eigenvectors of the covariance matrix, which is argued by its coincident eigenvalue and total concerted motion of the protein, is correlated with the protein function.71 The frequency of the eigenvectors with larger eigenvalue can usually represent the total concerted motion. The residues at the loop region of the pore and the binding pocket of the toxin showed high fluctuations in PCA analysis. The residues Asp110, Ala111, and Ser188 showed the highest fluctuations 5760


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Figure 6. Per-residue contribution of AQP2 toward total binding energy by the MMPBS method. The representation is given as (a) citrinin, (b) T2 toxin, and (c) ochratoxin-A. The negative energy indicates the high attractive energy between the protein and mycotoxins. The positive values indicate the repulsive energy at the binding site. Both positive and negative energies at the binding site disrupt the water flow through the channel and influence the functioning of the protein.

Table 1. Summary of Water Permeability Characteristics of Human AQP2 and Toxin-Bound AQP2a AQP2 AQP2_toxins

pd (10−14 cm3/s)

pf (10−14 cm3/s)

pf/pd

the net flow of water molecules along −z-direction

3.32 ± 0.75 1.08 ± 0.03

10.13 ± 0.07 10.58 ± 0.03

3.20 ± 0.70 10.05 ± 0.5

1007 52

Mycotoxin inhibition leads to the decreased flow of water through the channel.

a

hopping rate method to determine the effective free-energy profile opposing proton transport in bovine aquaporin-1.72,73 Burykin and Warshel estimated the free energy for transferring an excess proton to the interior of the b-Aqp1 pore using the protein−dipole Langevin−dipole method.74 The desolvation barrier opposing the intrusion of H+ into the narrow pore was compounded by a static barrier peaking at the NPA motifs, which opposes both the movement of protons and the reorientation of water molecules inside the pore. The static field is primarily due to the macrodipoles of two head-to-head α-helices converging at the NPA motifs. Our calculations of dipole moment and the electrostatic potential of the aquaporin-2 channel with mycotoxins conclude that the action of the toxin might lead to the proton conduction through the channel. Also, the interaction within the pore region created a net electrostatic field, which leads to the dysfunction of the channel. Further, superpositioning of the first and last trajectory frames and RMSF of PCA-based Cα atoms allowed us to discover that mycotoxins inhibit the aquaporin channel by a direct steric blockage, which also leads to the pore closure. The per-residue contribution of binding energy in the active sites revealed a strong affinity of the mycotoxins at the pore (Figure 6). Further, DFT calculations were used to confirm the high binding energy of mycotoxins (Figure S3 and Table ST1) with the surrounding residues, which was found to be consistent with the simulation results. There are two known mechanisms involved in the inhibition of the aquaporin channel: binding of molecules in the NPA motifs leading to the collapsing of the structure and blocking of the pores.72−74 3.2. Water Permeations through AQP2. To estimate the single-channel diffusive water permeability constants pd and pf, the number of water molecules that cross the channel in both positive and negative directions along the z-axis (±N) were calculated by the 100 ns molecular dynamics simulations. There are 69 and 109 water permeation events in the +zdirection and −z-direction, respectively. The number of water molecules that cross the channel per unit time is calculated by eq 2, and the diffusion permeability constants of water are

that were situated in the pore region, and the corresponding secondary structural changes are shown in Figure S2. The positioning of the residues Arg187, Phe48, His172, and Cys181 at the pore region plays a significant role as it controls the channel diameter (Figure 3). A significant change in the distances was noted between these residues on the first and last frames of the simulations. It was found that the interaction of mycotoxins pushed these residues inward and reduced the diameter of the pore. Accessibility of arginine and cysteine residues at the pore region and noncovalent interactions of molecules allowed them to create strong nonbonded interactions at the pore. Specifically, binding of the molecules induced rearrangement of the side chains of the aromatic arginine selectivity filter. The automated docking positioned mycotoxins inside the pore, coordinated by an extensive series of hydrogen bonds and hydrophobic interactions. Citrinin forms conventional hydrogen bonds with Arg85 and Ser2 residues. The π−alkyl stacked interaction with His80 and Ala12 along with several van der Waals interaction creates a strong electrostatic hub at the pore region. Likewise, OTA shows hydrogen bonds with Cys75 and Thr149 along with repulsive interaction with Asp150 and Glu151, whereas T-2 toxins showed more attractive interaction with several residues at the binding site. All of these hydrogen bonds persist during the simulation, and an average of four hydrogen bonds were formed through the simulation time, as shown in Figure 4c. Nonbonded interaction energies such as for van der Waals and electrostatic interactions play a significant role in the inhibition of AQP2. The OTA and T-2 toxin posses a high van der Waals interaction energy inside the binding cavity, whereas citrinin contributes the same or more electrostatic interaction energy (Figure 5). Interestingly, dipole moment and the electrostatic potential (ESP) of the ar/R region significantly reduced in the first and last frames. Previous studies show that the electrostatic forces in the pore region help aquaporin for the blockage of proton transport. Groot et al. combined nonequilibrium molecular dynamics simulations with stochastic proton jump using the quantum mechanically derived proton 5761


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Figure 7. (a) Modeled three-dimensional structure of AVPR2 receptor. (b) Ramachandran map of the optimized model, optimized model having 99.1% residues in the favored region, and all of the steric hindrances and bad contacts were removed by MD simulation. (c) Amino acid sequence obtained from UniProt with accession no. P30518.

convergence parameters of the I-TASSER’s structure assembly refinement simulations. Subsequently, the quality assessment of the modeled and refined protein models was performed by ψ/ϕ inspection of the Ramachandran plot obtained from PROCHECK analysis.77 To correct the stereochemistry and to eliminate bad contacts between protein atoms and structural water molecules, the models were solvated and subjected to constraint energy minimization followed by a 100 ns MD simulation in aqueous media. The conformation of the 100 ns simulation was obtained and subjected to PROCHECK analysis and found that more residues are confined to the core region of the Ramachandran plot (Figure 7b). Further, this structure was used for docking and molecular dynamics simulations to assess the effect of mycotoxins on AQP2 and AVPR2. Docking of toxin molecules in AVPR2 receptor was performed using Autodock, and the detailed procedure has been described elsewhere.78,79 Figures S5 and S6 show the interactions at the binding site; citrinin interacts with Trp99, Gln96, Phe105, Phe307, Val183, Trp193, Trp9, and Cys192 residues and forms hydrogen bonds with Cys192, Phe105, and Gln96. Ochratoxin-A shows hydrogen bonds with residues Arg137, Ser329, and Ser330, and π−sulfur and π−cation interactions with Met133 and Arg137 residues, whereas T-2 toxins form a hydrogen bond with Ser329 (Figure S6). Several distant residues interacting with the mycotoxins were added on condition that they were not at a distance longer than 4.5 Å. The hydrogen bonds formed at the binding site during the simulation time are given in Figure 7d, and the time evolution of secondary structural changes was calculated and is shown in Figure S7. At the end of the simulation, citrinin-bound AVPR2 has secondary structure content of 60.4% helix, 3.2% turn, and 36.4% coil. OTA-bound protein shows 59.8% helix, 6.5% turn, and 33.7% coil, whereas T-2-bound receptor shows 56.9%

computed using eqs 1 and 4. The ratio of osmotic to diffusive permeability constants related to the average occupancy number of water molecules in the channel as is calculated as pf/pd = N + 1,64,65 which are given in Table 1, and are found to be in agreement with the CTRW model64 occurring in a single file fashion. The water permeation of AQP2 coexisting with toxins showed a significant amount of reduction in the permeation events. There are 28 and 30 water permeation events in the +z-direction and −z-direction observed, respectively, and their permeability coefficients were calculated and are summarized in Table 1. The reduction of the permeation events indicates the functional damages induced by the mycotoxins. 3.3. Inhibition of AVPR2 by Mycotoxins. The role of arginine vasopressin receptor 2 is very crucial for the translocation of the AQP2 on the apical membrane. Inhibition of the receptor leads to the initial breakdown of the pathway involved in the AQP2 translocation. In this regard, it is essential to assess the effect of mycotoxins on the receptor protein. Unfortunately, there was no crystal structure available for the AVPR2 receptor. Hence, we modeled the receptor by obtaining the amino acid sequence from UniProt with accession no: P30518; the amino acid sequence and modeled proteins are given in Figures 7a and 6c; and the docked protein−ligand complex structures are shown in Figure S5. The homology model of arginine vasopressin receptor 2 (AVPR2) was constructed using I-TASSER,75 which implemented composite modeling approach that includes the identification of appropriate templates, reassembly of fragment structure, building of atomic models, and selection of the best model (Figure 7). The quality of the predicted receptor structure was assessed by confidence score,76 which is based on the significance of the threading template alignments and the 5762


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Figure 8. Histopathological changes of the kidney tissues: (a) normal glomerulus, (b) mesangial matrix expansion, (c) blood vessel congestion, and (d) hypercellularity. (e, f) Autolysis of the cells/tissues (see Section 3.3 for further descriptions).

helix, 5.4% turn, and 37.7% coil, indicating transformation from the initial percentage of 61.2% helix, 3.2% turn, and 34.2% coil and 1.3% 3-10 helix, respectively. Further, the PCAbased Cα RMSD calculations of the proteins showed regular peaks, which indicate the receptor instability (Figure S4). These conformational rearrangements may lead to the inactivation of the receptor, which prevents the expression of the AQP2 in the apical membrane. In summary, molecular docking and molecular dynamics simulations on aquaporin-2 and arginine vasopressin receptor 2 reveal the structural details at the atomic level. Docking provides the binding locations of the toxin molecules, and simulations provides dynamic information of the channel proteins. The structural changes in AVPR2 will lead to the inactivation of the receptor, as a result of which the expression of AQP2 will be altered. The equilibrium molecular dynamics simulation revealed reduction in permeation due to the structural changes induced by the toxin. Further, the histopathology of the mice kidney tissues was performed to evaluate the morphological changes induced by the toxin. RTPCR and WB used to identify the altered expression of the proteins, which is predicted by simulation. 3.4. Toxicity of Mycotoxins in the Renal System. We have evaluated the toxicity of mycotoxins in the renal systems of albino mice. Histological analysis was performed to see the morphological changes in the kidney and to identify possible damages induced by the administration of the mycotoxins. Further, the expression of AQP2 and AVPR2 was assessed using western blot and RT-PCR methods. 3.4.1. Histological Changes of the Kidney due to the Mycotoxin Administration. Histological analyses were performed to identify possible microscopic alternations of the kidney due to the administration of the mycotoxins. The cortex and medulla of the control samples were normal and a good number of glomerulus were seen in the section, and the highermagnified section shows delicate and thin glomerular capillary loops. The endothelial and mesangial cells are normal in

number, and no blood vessels congestion, Bowman’s capsules with the regular arrangement, and significant pathological changes were observed (Figure S8a−d and Figure 8a). However, the histology of the kidney which treated with individual and the combinations of toxins shows significant changes in the morphology. It shows degenerative changes in most regions compared to control; there was blood vessel congestion in all of the samples, indicating the features of various nephritis such as acute tubular necrosis (ATN), focal segmental glomerulosclerosis (FSGS), interstitial nephritis, lupus nephritis, and mesangial hypercellularity. The normal glomeruli and cells and the corresponding cortex tubules and medulla regions are shown at lower magnifications in Figures S8−S10. Figure 8b shows the mesangial matrix expansion in the glomeruli, blood vessel congestion, and the expansion of mesangial matrix manifesting diverse pathological conditions in the kidney. It may be due to the deposit of mycotoxins in the glomeruli or it may be the main reason for lupus nephritis. The sclerosis of the glomerulus supports the possibilities of the induced nephritis due to the toxins. Further, the death of many epithelial cells is an indication of the damage of the aquaporin and other related proteins because of the high expression of aquaporin in epithelial cells.80−82 Furthermore, the sections of the kidney show acute tubular necrosis along with interstitial lymphoplasmacytic infiltrates (Interstitial nephritis). In summary, the kidney of mice administrated with all toxins shows a nuclear loss and complete loss of normal architecture (autolysis), and the histological analysis reveals various possibilities other than nephrogenic diabetic insipidus. 3.4.2. Gene Expression and Immunoblotting of AQP2. The expressions of AQP2 mRNA were assessed by real-time PCR, and the results are depicted in Figure S11. The untreated and treated samples show almost threefold less expression in both AQP2 and AVPR2. Further, immunoblot analysis of membrane proteins from mice kidney cells with the polyclonal rabbit anti-AQP2 antibody recognized bands at 28 kDa. There was a higher level of expression in the untreated samples, 5763


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Figure 9. (a) Change of energy upon mutation; negative energy indicates the reduced stability, and positive energy indicates that mutation enhanced the stability of the channel. (b, c) Mutated residues location in the channel and mesh representing the suggested binding location.

Figure 10. Network diagram of aquaporin showing interdependence of residues. The residues Arg187, Gly180, His172, and Val169 correspond to the pore region/selectivity filter of the channel. The other residues connected with the main node influence the selection and transportation of the water molecules. Further, Val71, Val168, His66, and Gly64 situated at the cavity also interact with the water molecules in the channel.

Val71. It was found that the structural rearrangements of these residues will lead to the loss of function of the aquaporin channel, and mutation analysis shows higher changes of free energy associated with these residues. The second binding residues identified in the NPA region of the channel have also led to the loss of function. However, we focused on the residues at the pore region due to the inability of molecules to access the residues at the NPA motif. Further, we aligned the human AQP2 protein channel with those of model organism to understand the conservation of residues in the channel. Interestingly, almost 42 resides were preserved during the evolution from prokaryotes to eukaryotes and these residues were mutated with alanine to observe the changes in energy (Figure 9). The biological relevance of these residues depends

whereas the mycotoxin-treated samples showed significantly reduced expression levels or no expressions. Likewise, polyclonal rabbit anti-AVRP2 antibodies revealed a similar expression level as observed for aquaporin-2 membrane channels. Hence, this establishes the effect of toxin-induced damages on the aquaporin-2 membrane protein channels and arginine vasopressin receptors. 3.5. Interdependence of the Residues and Evolutionary Conservation of AQP2. To understand the role of individual amino acids toward the stability of the AQP2, we mutated the residues and calculated the changes of stability with respect to free-energy changes. The mycotoxin interactions revealed the unique binding locations that consist of His172, Arg187, Val168, Gly180, Cys181, Gly64, His66, and 5764


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4. CONCLUSIONS The present study revealed the structural and functional changes of the aquaporin-2 channel upon the interaction of mycotoxins such as citrinin, ochratoxin-A, and T-2 toxin. The molecular docking provided an initial conformation for the protein−ligand complexes. The hydrogen bond and hydrophobic interaction details obtained from the docking assisted understanding of the strong affinity of the toxin with AQP2. Further, the transport properties and conformational dynamics of AQP2 have been studied using extensive unbiased MD simulations. We have shown that the presence of the mycotoxins significantly reduced the permeation of water molecules across the AQP2 channel. The water molecules encounter a significant obstacle around the ar/R region, which is the narrowest part of the channel. The binding free-energy calculation by the MM-PBSA method shows that the electrostatic interactions at the pore region are the primary reason for the barrier and the creation of an electrostatic field at the pore region. The interaction of toxins with the pore region causes a conformation rearrangement, which leads to the closure of the channel. The critical residues (His172, Arg187, Val168, Gly180, Cys181, Gly64, His66, and Val71) taking part in the inhibition mechanism were identified, and the DFT calculations confirmed that these residues form a strong interaction at the binding site. The residue-wise evolutionary coupling analysis also implicates the importance of these residues in the channel. The histopathological analysis of mycotoxin-treated mice shows significant changes in the glomerulus and associated tissues. Western blot along with RT-PCR results reveals the low-level expression of AQP2 channel in the kidney, which reveals the possibilities of nephrogenic diabetes insipidus and nephrotoxicity of the mycotoxins. In addition, the inhibition of AVPR2 receptors blocks the G-protein-mediated protein kinase A pathways, which leads to failure in the translocation of AQP2 to the apical membrane. Hence, our study implicates that the interaction of mycotoxins with AQP2 and AVPR2 leads to the inhibition of both protein functions. The in vivo studies help to understand the possibilities of NDI and various pathological conditions caused by mycotoxins. These data may help to design targeted specific drugs or therapeutic molecules against AQP2.

on the positioning of amino acids in the channel; experimental studies show that the mutation of serine residues in different regions (148, 229, 231, 244, and 256) results in failure of the aquaporin to express in the apical membrane as a response to the vasopressin.83 Verkman et al. observed the mutation at L22V in a female patient with symptoms of NDI from infancy.84 Marr et al. identified seven mutations in the gene encoding AQP2 (AQP2-L28P, AQP2-A47V, AQP2-V71M, or AQP2-P185A) that cause recessive NDI, and they further reported that all AQP2 missense mutations that cause autosomal recessive NDI are class II mutations; however, some of these mutants appear to be able to function as water channels.83 Further, the residues Arg187, Gly180, His172, and Val169 correspond to the pore region/selectivity filter of the channel. The other residues which connected with the main node influence the selection and transportation of the water molecules. Further, Val71, Val168, His66, and Gly64 situated at the cavity that interacts with the water molecules in the channel. The critical residues identified in this study are located inside the channel surface where the water conduction occurs. Statistical methods such as mutation and statistical coupling analysis provided the interdependency of the residues of the AQP2 channel; the SCA matrix corresponding to human AQP2 with other water transported proteins in the UniProt database is provided in the Supporting Information. Figure S12 shows a clustering tree based on χ2 scores weighted by phylogeny with the focus on the residues identified from mycotoxins interactions and conserved residues during the evolution that are clustered together.85−87 Remarkably, the residues identified from the mycotoxins interaction and point mutations lie within the same cluster. The circular diagram with the closest relationships to the identified residues is shown in the Supporting Information (Figure S13), and the corresponding network diagrams are given in Figure 10. It can be seen that residues identified through MCA, SCA, and mycotoxins interactions were located inside the channel and near to selectivity filter. Further, the network diagrams of these residues showing higher interdependence in the aquaporin channels, including Mus musculus, Bos taurus, Escherichia coli, Komagataella phaffii, and Homo sapiens, are given in Figures S14−S18. The residue Arg187 originates from the node that shares the residues Pro208, Trp202, and His177, whereas the residue His172 was observed in a larger node with 18 other amino acids, including Gly180 and Leu141. Further, the complete covariance was identified between the residues Gln93, Ala186, Ala70, Ala66, Ser63, Val71, Trp202, Pro208, Arg187, Gly49, Gln93, Tyr89, Ser63, His66, Gly27, Ala15, Gly64, Gly180, and Leu141. Remarkably, these residues were included in the mycotoxin interaction site, such as His172, Arg187, Val168, Gly180, Cyc181, Gly64, His66, and Val71, which showed a complete covariance. Similarly, other related residues in the interaction site lie in the relative chain of the phylogenic chart. In addition, we adapted the evolutionary couplings (ECs) model to predict the contact within protein, which has evolutionary importance (Figure S19). EV coupling analysis88 was performed over the protein sequences of organisms such as Dictyostelium discoideum, Drosophila melanogaster, Arabidopsis thaliana, Escherichia coli, Rattus norvegicus, and Mus musculus along with human aquaporin-2. As a result, we propose a unique binding locations/target (His172, Arg187, Val168, Gly180, Cys181, Gly64, His66, and Val71) for mutations or to develop inhibitors for the aquaporin-2 channel.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.9b03829.

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Supporting data for MD simulations (Figures S1 and S2); additional images for in vivo studies (Figures S8− S10); and additional figures for the network analysis (Figures S11−S18) (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (N.M.). *E-mail: [email protected] (P.K.). ORCID

Nikhil Maroli: 0000-0001-7591-9853 Author Contributions

N.M. designed work and performed the simulation. A.J. and R.R.M. performed in vitro studies. P.K. supervised the work, 5765


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and K.K. provided the facilities as director of the center. N.M. prepared the manuscript, and A.J., R.R.M., and P.K. approved the final version. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. S. Kannan, Department of Zoology, Periyar University, Tamil Nadu, India, for the support toward the animal study, and Balu Bhasuran for his help in network diagrams. This work was supported by the Defence Research and Development Organisation (DRDO), Ministry of Defence, India [grant number DLS/86/50011/DRDO-BU Centre/ 1748/D (R&D) dtd 4th J].

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