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Phosphorylation of Ser-180 of rat aquaporin-4 showsmarginal affect on regulation of water permeability:molecular dynamics studyRuchi Sachdeva a & Balvinder Singh aa Bioinformatics Center , CSIR-Institute of Microbial Technology, Council of Scientific andIndustrial Research , Sector 39A, Chandigarh , 160036 , IndiaPublished online: 07 May 2013.

To cite this article: Ruchi Sachdeva & Balvinder Singh (2013): Phosphorylation of Ser-180 of rat aquaporin-4 shows marginalaffect on regulation of water permeability: molecular dynamics study, Journal of Biomolecular Structure and Dynamics,DOI:10.1080/07391102.2013.780981

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Phosphorylation of Ser-180 of rat aquaporin-4 shows marginal affect on regulation of waterpermeability: molecular dynamics study

Ruchi Sachdeva and Balvinder Singh*

Bioinformatics Center, CSIR-Institute of Microbial Technology, Council of Scientific and Industrial Research, Sector 39A, Chandigarh160036, India

Communicated by Ramaswamy H. Sarma

(Received 15 November 2012; final version received 24 February 2013)

Water permeation through rat aquaporin-4 (rAQP4), predominantly found in mammalian brain is regulated by phosphor-ylation of Ser-180. The present study has been carried out to understand the structural mechanism of regulation of waterpermeability across the channel. Molecular dynamics (MD) simulations have been carried out to investigate the structuralchanges caused due to phosphorylation of Ser-180 in the tetrameric assembly of rAQP4 along with predicted C-terminalregion (255–323). The interactions involving opposite charges are observed between cytoplasmic loops and the C-terminal region during MD simulations. This results in movement of C-terminal region of rAQP4 towards thecytoplasmic mouth of water channel. Despite this movement, there was a gap between C-terminal region and cytoplas-mic mouth of the channel through which water molecules were able to gain entry into the channel. The interactionsbetween C-terminus and loop D of neighboring monomers in a tetrameric assembly appear to prevent the completeclosure of cytoplasmic mouth of the water channel. Further, the rates of water permeation through phosphorylated andunphosphorylated rAQP4 have also been compared. The simulation studies showed a continuous movement of water ina single file across pore of unphosphorylated as well as phosphorylated rAQP4.

Keywords: rAQP4; regulation; water permeability; aquaporin; phosphorylation

Introduction

Aquaporins have been primarily assigned the function ofwater transport across the cell membrane, thereby con-tributing to the maintenance of proper fluid balanceacross cells, especially in case of red blood cells, lens,brain, and kidney cells. In addition to water, aquaporinstransport small, neutral solutes such as ammonia(Saparov, Liu, Agre, & Pohl, 2007), glycerol (Hara-Chikuma & Verkman, 2005), urea (Ishibashi et al.,1994), etc. The transport of water across aquaporinchannel is regulated either by gating or by trafficking ofaquaporins being targeted to different membranes.Changes in pH, divalent cation concentration, osmolality,and phosphorylation of serine or threonine residues are afew signals through which aquaporins are gated. In caseof AQP0, Nemeth-Cahalan and Hall (Nemeth-Cahalan &Hall, 2000) have demonstrated that decrease in externalpH increases water permeability of AQP0 by three folds.Gonen, Sliz, Kistler, Cheng, and Walz (2004) hassuggested that protonation/deprotonation of His-40 andHis-66 is likely to affect the water permeability of

AQP0. Phosphorylation induced gating of aquaporin isanother mechanism observed in SoPIP2;1, a spinachaquaporin. Molecular dynamics (MD) simulations ofSoPIP2;1 by two different studies show that phosphory-lation of different serine residues at position 115 and 188results in displacement or conformational change of loopD followed by channel opening (Nyblom et al., 2009;Tornroth-Horsefield et al., 2006). Also, in case of yeastAqy1, Ser-107 in loop B has been identified as aputative phosphorylation site (Fischer et al., 2009). It isbeing speculated that phosphorylation of this Ser-107residue may displace Tyr-31 from the channel, therebyleading to an open conformation. There is a wealth ofstructural data available for phosphorylated inducedopening of the aquaporin channel e.g. in case ofSoPIP2;1 and Aqy1. However, there is little dataavailable about mechanistic details of closure of theaquaporin channel due to phosphorylation.

Aquaporin-4 (AQP4) is a predominant water channelin the mammalian brain (Jung et al., 1994) and isexpressed in endfeet of astrocytes surrounding the blood-

*Corresponding author. Email: bvs@imtech.res.in

Journal of Biomolecular Structure and Dynamics, 2013http://dx.doi.org/10.1080/07391102.2013.780981

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brain barrier (Amiry-Moghaddam & Ottersen, 2003) andin the collecting duct of kidneys (Hasegawa, Zhang,Dohrman, & Verkman, 1993). A shorter spliced variant ofAQP4, M23, assembles into orthogonal arrays, whereas alonger spliced variant, M1 has little propensity to formarrays. A putative protein kinase A phosphorylation site,Ser-111 has been found to be involved in regulation ofarray formation in rat AQP4 (rAQP4) (Silberstein et al.,2004; Van Hoek et al., 2009). Expression of M23-rAQP4Ser111Glu mutant mimicking the phosphorylation resultedin larger arrays than wild type (Silberstein et al., 2004).AQP4 is involved in brain water accumulation that under-lies the formation of cerebral edema (Amiry-Moghaddamet al., 2004; Papadopoulos & Verkman, 2005), thus mak-ing the regulation of this channel critical for life. Zelenina,Zelenin, Bondar, Brismar, and Aperia (2002) have shownthat activation of protein kinase C has resulted in decreasein water permeability of rAQP4 expressed in the basolat-eral membrane of kidney epithelial cells. The structure ofAQP4 has been determined by electron crystallography ofdouble–layered, two-dimensional (2D) crystals (Hiroakiet al., 2006). Phosphorylation of Ser-180 has beenimplicated in protein kinase C and dopamine-dependentdecrease in rAQP4 water permeability due to gating(Zelenina et al., 2002). This effect was not observed whenSer-180 was mutated into alanine. Since Ser-180 ispresent near the cytoplasmic mouth of the channel, phos-phorylation of this residue is likely to increase chances ofits interactions with C-terminal region of rAQP4. It hasbeen speculated by Hiroaki et al. (2006) that phosphory-lated Ser-180 may, in turn interact with positively chargedresidues present in the C-terminus (Lys-259, Arg-260,Arg-261) of rAQP4. Blocking of the cytoplasmic mouthof the channel by this interaction may explain gating ofrAQP4 channel due to phosphorylation (Hiroaki et al.,2006). However, this proposed mechanism of rAQP4gating needs to be established.

In the present study, we have carried out MD simulationstudies on the transport of water molecules across rAQP4channel and attempted to investigate the mechanistic detailsof closure of rAQP4 induced by phosphorylation, for thefirst time. We have modeled C-terminal region of rAQP4that was missing in the electron crystallographic structure tostudy its role in the regulation of water transport via gating.The detailed interactions of phosphoserine-180 that couldoccur with positively charged residues of the C-terminalregion of rAQP4 are presented. The effects of such intramo-lecular interactions on water transport across rAQP4 channelper se are discussed in this study.

Methods

Homology modeling

Structural information was not available in the PDB file(PDB ID 2D57) for residues 1–30 at N-terminus and

255–323 at C-terminus of rAQP4 (Hiroaki et al., 2006).Amino acid residues from 255 to 267 were modeledchoosing the crystal structure of bovine AQP1 (PDB IDcode 1J4N) (Sui, Han, Lee, Walian, & Jap, 2001) astemplate. SH3 domain of T cell Adapter protein ADAP(Heuer, Kofler, Langdon, Thiemke, & Freund, 2004) hasbeen predicted to be the best template for C-terminalregion of rAQP4 by I-TASSER (Zhang, 2008) with 27%of sequence identity in threaded aligned region. Thus,rAQP4 containing the C-terminal region from 31 to 323(i.e. for amino acid residues 31–254, 255–267 and 268–323) was modeled using multiple templates: (rAQP4(PDB ID: 2D57), bovine aquaporin-1 (PDB ID: 1J4N),and SH3 domain of T cell Adapter protein (PDB ID:1RI9), respectively) with the help of Modeller9v7 (Sali& Blundell, 1993). Sequence alignment used for buildingmodel (31–323) of rAQP4 is given in supplementaryFigure 1. Amino acid residues 31–254 present in therAQP4 structure were kept fixed during model building,while the C-terminal region of rAQP4 from residues255 to 323 was allowed to be optimized during theconstruction of models. Further, secondary structure ofC-terminal region (255–323) of rAQP4 was predicted byPSIPRED (McGuffin, Bryson, & Jones, 2000) accordingto which a region of 18 residues showed propensity toform helix and another three regions spanning over range

Figure 1. Structure of rAQP4 along with modeled C-terminalregion (255–323). The C-terminal helix contains 259KRR261residues while Ser-180 is located on loop D.

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of 2–6 residues have shown propensity for β-strands.Top ten models with lowest DOPE score as predicted byModeller were initially selected for C-terminal region ofrAQP4. A few of these predicted models were rejectedthat were found to have their atoms coming too close tothose of same monomer or neighboring monomers andeven lipid molecules after constructing the tetramericassembly of rAQP4. These models were further filteredbased on the comparison between secondary structure ofC-terminal region of rAQP4 predicted by PSIPREDand one obtained from the predicted tertiary model ofC-terminal region of rAQP4. Two models namedrAQP4-I and rAQP4-II were selected for further studies.Tetramer of the resulting model of rAQP4 containing theC-terminal region was constructed using transformationmatrix provided with the rAQP4 structure using VMD(Humphrey, Dalke, & Schulten, 1996).

Preparation of system

In order to study the structural changes caused uponphosphorylation of rAQP4, a phosphate group wasattached to hydroxyl group of Ser-180 in both modelsrAQP4-Ip and rAQP4-IIp. Histidine residues wereprotonated at the ɛ position. Each model was embeddedin a 100� 100Å2 patch of POPC lipid bilayer built byMEMBRANE plugin of VMD (Humphrey et al., 1996).Surface tyrosine residues were used to adjust the positionof rAQP4 model along the membrane normal. Lipidmolecules with heavy atoms closer than 0.8Å to anyatom of the protein were removed. The systems werethen placed in a TIP3P (Jorgensen, Chandrasekhar,Madura, Impey, & Klein, 1983) water box of size99Å� 99Å� 100Å using the SOLVATE plugin ofVMD. Water molecules lying in the membrane hydro-phobic region were removed and there is at least a 20Ålayer of water molecules surrounding the exposed partsof rAQP4 models. Total number of water molecules inunphosphorylated (rAQP4-I) and phosphorylated(rAQP4-Ip) systems were 21,022 and 21,021, respec-tively. Net charge of the systems was made zero by add-ing counterions using AUTOIONIZE plugin of VMD.Na+/Cl� ions were added in order to set the ionic con-centration as 100mM.

Simulation

Phosphorylated and unphosphorylated rAQP4 systemswere minimized in three stages: initially, only water andions were minimized for 8000 steps using conjugategradient method, while keeping the protein and lipidmolecules fixed. While still keeping protein atoms fixed,the systems were further minimized for 15,000 steps ofconjugate gradient. Later, all atoms were minimized for20,000 conjugate gradient steps. The minimized systemswere first simulated for 20 ps during which only the lipidtails were allowed to melt at 450K in NVT ensemble in

order to introduce appropriate disorder of a fluid-likebilayer keeping all other atoms, i.e. water, lipid headgroups, ions, and protein fixed. First equilibration wasperformed for 40 ps at 310K with constraints on proteinatoms, lipid head groups, lipid tails, water, and ionsusing force constants 12, 10, 5, 3, and 3 kcal/mol/Å2,respectively. It was followed by another run for 30 ps,where constraints were reduced by taking force constants8, 7, 3, 2, and 2 kcal/mol/Å2, for protein atoms, lipidhead groups, lipid tails, water, and ions, respectively.Force constants were further reduced to 4, 4, and 2 kcal/mol/Å2, on protein atoms, lipid head groups, and lipidtails, respectively in the next round of equilibration of30 ps while removing the constraints on water and ions.Then, simulation was carried out for 50 ps, where forceconstants used were 4, 2, and 1 kcal/mol/Å2, on proteinatoms, lipid head groups, and lipid tails, respectively.During MD simulations of rAQP4 models, it wasobserved that side chain of Arg-216 of ar/R site used toblock the channel by protruding into the pore. It wasfound to adopt two conformations in which the dihedralangle, χ4 exhibited an average value of about �100° and�170°, respectively, with the channel transiently blockedin the latter conformation. Therefore, χ4 of Arg-216 wasconstrained to �100° (force constant 8 kcal/mol/Å2,)during subsequent equilibration runs. All these simula-tions were carried out in NVT ensemble. The systemswere further equilibrated at 310K and at a constant pres-sure of 1 atm (NPT) for 2850 ps, while constraining thedihedral angle, χ4 (Cγ-Cδ-Nɛ-Cζ) of Arg-216 and rest ofthe atoms of protein with a force constant of 8 and4 kcal/mol/Å2, respectively. This was followed by 6 nsequilibration with reduced constraints on protein (3 kcal/mol/Å2) and on χ4 of Arg-216 (5 kcal/mol/Å2). Later,constraints were released and the systems were furthersimulated for 1 ns. Finally, simulation has been carriedout on phosphorylated as well as unphosphorylated mod-els for 30 ns in production run. All MD simulations werecarried out using NAMD software package (Phillipset al., 2005) with the CHARMM27 parameter set(MacKerell et al., 1998). Simulation was carried out at1 atm using the Langevin piston at a constant simulationtemperature of 310K along with periodic boundary con-ditions and time step of 2 fs. Bonds between each of thehydrogen and its mother atom were constrained duringsimulation using SHAKE algorithm (Ryckaert, Ciccotti,& Berendsen, 1977). Electrostatic interactions were cal-culated using the particle-mesh Ewald method (Essmannet al., 1995) and Van der Waals interactions were cut offby using a switching function from 10 to 12 Å. Thecoordinates were saved at every 2 ps. All the simulationswere performed on 16 Quad core AMD Opteron proces-sor cluster. Osmotic permeability (pf) of water channelwas calculated as mentioned in Zhu, Tajkhorshid, andSchulten (2004).

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Results and discussion

Modeling rAQP4 along with C-terminal region

The structural data were missing in the PDB file (2D57)for first 30 amino acid residues in the N-terminus andlast 69 residues in C-terminus of the rAQP4. Therefore,C-terminal region spanning from 255 to 323 amino acidresidues has been modeled using Modeller. Two modelswith lowest DOPE score were selected based on criteriaas mentioned earlier. These models have similar second-ary structure content. In one of the models (rAQP4-I), astretch of 10 residues and 2 stretches of 3 residues arepredicted to have α-helix and β-strands, respectively. Thepredicted C-terminal region of rAQP4 is mostly coiledcoil as shown in Figure 1. The side chain of two argi-nines among KRR of C-terminal region was pointingtowards the oxygen of side chain of Ser-180 in themodel, whereas Lys-259 was in opposite orientation(Figure 1). None of the residues of C-terminal region isin contact with transmembranous part of rAQP4. TherAQP4-I model contained 99% of residues in allowedregion of Ramachandran map. The z score indicatingoverall model quality computed by ProSA (Wiederstein& Sippl, 2007) was found to be 0.14 which is within therange of scores typically found for experimentallydetermined protein structures of similar size. The secondmodel (rAQP4-II) was found to have 96% residues inallowed region of Ramachandran map and a z score of0.41. Stereochemical quality of final simulated structureshas been verified by scores of Verify 3D (Luthy, Bowie,& Eisenberg, 1992) and by plotting Ramachandran maps(Supplementary Figure 2). The scores of Verify 3D havebeen found to be within the range of good quality struc-tures for electron diffraction structure (SupplementaryFigure 3) and for all monomers of unphosphorylated

(Supplementary Figure 4) and phosphorylated simulatedstructures of rAQP4. More than 99% amino acid residuesare in the allowed region of Ramachandran maps forthese simulated structures of rAQP4. The z-scoresobserved for electron diffraction structure and modelsimulated structures have been �3.67 and �0.95, respec-tively as computed by PSVS method (Bhattacharya,Tejero, & Montelione, 2007) while G-factors, ascomputed by PROCHECK (Laskowski, MacArthur,Moss, & Thornton, 1993) for these structures have been�0.62 and �0.16, respectively. The folding free energyof electron diffraction structure and simulated structure(averaged per monomer) is comparable with their valuesbeing �227.2 and �218.2 kcal/M, respectively. On theother hand, solvation free energies have been �9.6 and�176.7 kcal/M, respectively for electron diffraction andsimulated structures. Much lower solvation free energyof simulated model structure may be attributed to thepresence of C-terminal region of rAQP4 in thecytoplasm, being exposed to the solvent as the same ismissing in electron diffraction structure, 2D57.

Intrinsic disorder of sequence of rAQP4 waspredicted using different methods: DisProt (Obradovicet al., 2003), disEMBL (Linding et al., 2003), andPrDOS (Ishida & Kinoshita, 2007). The results of thesemethods showed that first 10 N-terminal residues andamino acid residues from 255 to 323 in the C-terminalregion of rAQP4 have greater disorder probability ascompared to the transmembranous region. Disorder prob-ability was found to be lower in the regions – 255–268and 291–305, which contain a helix- and beta-strand inthe predicted structure, respectively.

Simulation and structural stability of rAQP4

Conformational changes in rAQP4 models weremonitored for 31 ns of MD simulation by computingbackbone and heavy atoms root mean-square deviation(RMSD) relative to the energy minimized structures.RMSD of backbone atoms of residues from 31 to 254(excluding the modeled C-terminal region) of phosphory-lated, rAQP4-Ip, and unphosphorylated, rAQP-I wasfound to be below 2.5Å indicating little conformationalchange due to phosphorylation (Figure 2). It wasobserved that RMSD of the C-terminal region only i.e.residues 255–323 was found to vary up to 16 and 13Åin unphosphorylated and phosphorylated rAQP4-I,respectively (Figure 2). Both rAQP4-I and rAQP4-Iptetramer assembly exhibited overall high RMSD due toavailability of more space for movement of C-terminalregion as compared to the aquaporin region from 31 to254 residues, which is mostly surrounded by lipid mole-cules of the bilayer. Root mean square fluctuations(RMSF) have been computed for C-terminal region(i.e. residues 255–323) of all the monomers of unphos-phorylated and phosphorylated rAQP4 during 30 ns of

Figure 2. RMSD of residues 31–254 of rAQP4-I (red) andrAQP4-Ip (black) in tetramer assembly of rAQP4-I during MDsimulations. RMSD of C-terminal region alone (i.e. residues255–323) was found to be low for rAQP4-Ip (green) than thatof rAQP4-I model (blue).

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dynamics (Supplementary Figures 5 and 6). Amino acidresidues from 255 to 265 show fluctuations less than 4Ådue to presence of a stable helix in all the monomers ofrAQP4. The amino acid residues (265 onwards) showhigher rms fluctuations as these adopt coiled coil struc-ture except for a small region around 290 ± 5. The aminoacid residues in this region have been predicted to forma beta-strand in the modeled structure of rAQP4. Further,higher RMS fluctuations for some of the amino acidresidues of C-terminal region could be due their shortlived interactions with cytoplasmic loops of the rAQP4.RMSF are very high for five amino acid residues at theC-terminal end of most of the monomers during 30 nsdynamics. These observations are in accordance with thepredicted disorder among these residues of C-terminalregion of rAQP4.

Radius of gyration (Rgyr) was computed for theC-terminal region of rAQP4 and it was found that itsvalue remained near 14Å or less in all the monomersexcept monomer D where it increased to 15Å and

became stable during the simulations. Despite overallhigh RMSD, C-terminal region remained compact duringsimulation of rAQP4-I and rAQP4-Ip. The region (fromresidues 253 to 256) linking C-terminal region withtransmembrane parts of rAQP4 showed movementduring simulation, resulting in displacement of wholeC-terminal region with respect to its initial position. Thisis likely to account for high RMSD observed in case ofC-terminal region.

Phosphoserine-180 interacts with positively chargedresidues

Distances were monitored between side chain oxygen ofSer-180 and the residues that lie within 15Å of Ser-180during the simulations of rAQP4-I and rAQP4-Ip.In case of monomers A and C, the distance betweenLys-109 and phosphoserine-180 decreased to 4Å andstabilized during MD simulations (Figure 3(A), (C), and(E)). Interaction between Lys-109 and phosphoserine-180was absent in monomer B. Monomer A revealed a close

Figure 3. Inter atomic distances in unphosphorylated (shown in red) and phosphorylated (shown in black) rAQP4-I during MDsimulations. Distance between Oγ of Ser-180 and (A) Nζ of Lys-109 in monomer A, (B) Nζ of Lys-311 in monomer A and (C) Nζ ofLys-109 in monomer C. Snapshots of MD simulations showing interaction between phosphoserine-180 of loop D (magenta) (D) withLys-109 and Lys-311 in monomer A and (E) with Lys-109 in monomer C. 259KRR261 residues (orange colored spheres) are locatedaway from phosphoserine-180.

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association between amino group of Lys-311 andphosphoserine-180 (Figure 3(B) and (D)). Near-neighborinteraction was observed between oxygen atom ofphosphoserine-180 and nitrogen of Lys-181 in all themonomers except monomer A. The side chains of theseneighboring residues came near to each other for shortintervals before moving away during MD simulations.This was repeated in all the monomers for different timeintervals in MD simulations. During last 6 ns, nitrogen ofHis-300 was in close contact with phosphoserine-180 inmonomer C. The interaction between phosphoserine-180and Lys-181 was replaced with Lys-263 after 10 ns ofsimulations in monomer D. At 12 ns, Lys-109 camecloser to phosphoserine-180 and was observed to partici-pate in bifurcated interactions with phosphoserine-180along with Lys-263 (Figure 4(A), (B), and 4D)). Itmay be noted that this Lys-263 is located on the helixof predicted C-terminal region of the rAQP4. MD

simulation of SoP1P2;1 showed similar interactionbetween phosphoserine-188 of loop D and Lys-270 ofC-terminus (Nyblom et al., 2009). Arg-260, a part of259KRR261 in the helix on C-terminal region was ableto establish a stable interaction with phosphoserine-180within 2 ns of simulation in monomer B (Figure 4(C)and (E)). Amino acid residues-259KRR261 belonged tostrongly predicted helical region in rAQP4 from residues258 to 265 as predicted by PSIPRED as well. Thishelical C-terminal region of rAQP4 showed sequencealignment with corresponding helical region in thestructure of bovine AQP1 (240–247) (Sui et al., 2001).MD simulations of rAQP4 modeled (from residues 31 to267) with bovine AQP1 as a template has shown similarinteraction between phosphoserine-180 and KRR regionof the C-terminal helix (data not presented). This kind ofinteraction has also been thought of as a possibility byHiroaki et al. (2006) with a speculation that this

Figure 4. Inter atomic distances of unphosphorylated (shown in red) and phosphorylated (shown in black) rAQP4-I as a function oftime. Distance between Oγ of Ser-180 and (A) Nζ of Lys-109 in monomer D, (B) Nζ of Lys-263 in monomer D and (C) NH2 ofArg-260 in monomer B. (D) MD snapshot shows interaction between phosphoserine-180 and Lys-109 located on cytoplasmic loop aswell as Lys-263 of C-terminal helix of monomer D. (E) Phosphoserine-180 of loop D (magenta) is involved in ionic interaction withArg-260 (orange colored spheres) of monomer B during MD simulations.

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interaction may block cytoplasmic mouth of the waterchannel after phosphorylation. The C-terminal helix inrAQP4-Ip is positioned on the same side of cytoplasmicmouth, where loop D containing Ser-180 is locatedthroughout MD simulations. Thus, the residues, Lys-109and Lys-181, present on the cytoplasmic loops and thoselying in the C-terminal region i.e. Arg-260, Lys-263,His-300, and Lys-311 interact with phosphoserine-180during the MD of rAQP4-Ip. The electrostatic changesassociated with C-terminal region of rAQP4 have beenobserved for phosphorylated and unphosphorylatedrAQP4. Electrostatic surface of C-terminal region ofrAQP4 is mostly negatively charged interspersed withneutral and positive charges. Phosphorylation of Ser-180does show accumulation of negative charge around this

region (Supplementary Figure 7-above) due to phospory-lation. However, interactions between phosphorylatedSer-180 and Arg-260, Lys-263 occur on the edge of themouth of aquaporin (in monomer A) neutralizing someof the negative charge at phosphorylated Ser-180.Similar interactions have been observed in the monomerD (Supplementary Figure 7-below) and other monomersof rAQP4. This redistribution of surface charge afterphosporylation may not have much effect on the move-ment of water across the channel due to the availabilityof approach of water molecules to mouth of channel. Inaddition to these residues, other amino acids such asLys-295 and Arg-284 were implicated in the interactionsobserved during simulations of second model of rAQP4(rAQP4-IIp). None of the residues in the C-terminal

Figure 5. (A) Glu-288 on C-terminal region of monomer A (yellow) interacts with Lys-259 and Lys-263 present on C-terminus helix ofmonomer C (green) of rAQP4-Ip. (B) Phosphoserine-180 from monomer A (yellow) is in close contact with Arg-260, a part of C-terminal helix of monomer C (green).

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helix of rAQP4-IIp were involved in interactions duringthe simulations.

In unphosphorylated model, Arg-260 was found to beinvolved in bifurcated interactions with Ser-180 andGlu-318 with former interaction getting disrupted duringlast 6 ns of simulation in monomer C only. However,Ser-180 was in close proximity of backbone oxygen ofGlu-318 of C-terminal region only throughout the simula-tions. Ser-180 failed to make an interaction with any of thepositively charged residues in C-terminal region as evidentfrom the large distances between these in other monomers(Figures 3 and 4). However, a few interactions such asbetween Arg-108 of cytoplasmic loop and Glu-264 ofC-terminal region in monomer D; between Lys-109,Lys-181, and Glu-264 of C-terminal region of monomer Bwere observed. The latter set of interactions was lost after5 ns of simulations. Instead, C-terminal helix was found tocome near to side chains of Thr-183 and Lys-181 of loop Dand Phe-266, suggesting hydrophobic interactions.Monomer A of rAQP4-I did not reveal any close contactbetween cytoplasmic loop and C-terminal region.

Interactions between neighboring monomers

Inter-monomer interactions were observed eitheramong C-terminal regions of neighboring monomersor C-terminal region of one monomer and loop Dof another monomer in the tetrameric assembly ofrAQP4-Ip. The side chain atoms of Lys-263 present onC-terminal helix of monomer C and Glu-288 belongingto C-terminal region of monomer A of rAQP4-Ip formeda salt bridge during MD simulations (Figure 5(A)). At28 ns, Lys-263 was replaced by Lys-259, also a part ofC-terminal helix of monomer C in this interaction withGlu-288, suggesting that both these residues are equallyprobable to come near to Glu-288 (Figure 5(A)).Towards the end of simulation, Arg-260 of monomer Cand phosphoserine-180 on loop D of monomer A camein close proximity (Figure 5(B)). Interactions were alsoobserved between Lys-259 present in C-terminal helix ofmonomer A and Glu-290 and Asp-184 of monomer D asindicated by decrease in their distances to less than 7 Å.Similar to our findings, MD simulations of SoPIP2;1 byNyblom et al. (2009), have revealed the formation of asalt bridge between Lys-270 of C-terminus from onemonomer and Asp-191 present in loop D of neighboringmonomer that enabled the release of loop D from theclosed state. In the second model of phosphorylatedrAQP4 (rAQP4-IIp), Glu-288 of monomer C was foundto interact with Gln-286 and Asn-283 of monomer A atdifferent time point and for different durations during theMD simulations. No interactions were observed betweenamino acids of loop D of a monomer with C-terminalregion of another monomer in the simulation of thismodel. The interactions involving amino acid residue oftransmembraneous region of rAQP4-Ip, Tyr-248 of TM

helix-6 with Asp-179 of loop D of same monomer andHis-90 of loop B of adjacent monomer were observed.Such interactions between loop D and loop B have alsobeen reported in an electron diffraction structure ofAQP4S180D mutant that mimics the phosphorylated Ser-180 (Mitsuma et al., 2010). In unphosphorylated model,rAQP4-I, Arg-260 present on C-terminal helix of mono-mer B was involved in bifurcated ionic interactions withAsp-291 and Glu-290 located in C-terminal region ofmonomer C. No close contact between C-terminal helixand loop D of other neighboring monomers wasobserved in the unphosphorylated rAQP4-I.

Flow of water across rAQP4

Water molecules were observed to enter both channelentrances after removal of constraints in MD simulations.There was a single file of water molecules acrosschannel in all the monomers of both unphosphorylatedand phosphorylated states of rAQP4. Bipolar orientationof water molecules in two halves of the channel wasseen during MD simulations of rAQP4 as observed inother studies carried out on aquaporins (de Groot &Grubmuller, 2001; Tajkhorshid et al., 2002). The flow ofwater molecules was occasionally disrupted as side chainof Arg-216 of ar/R site was found to interact with back-bone carbonyl oxygen of Ala-210 and block the channelfor short time interval. Similar to this, crystal structureof Escherichia coli AqpZ has revealed two distinctconformations of Arg-189 of ar/R site (Jiang, Daniels, &Fu, 2006). In one monomer, Arg-189 side chain pointstoward the periplasmic vestibule whereas in othermonomer, it bends over to form a hydrogen bond with

Figure 6. Top view (cytoplasmic side) of rAQP4 shows thatloop D (magenta) remains away from the mouth of waterchannel due to interaction between phosphoserine-180 of loopD and Lys-263 of C-terminal helix (magenta). Side chains ofamino acid residues on the periplasmic loops are shown aslines. Some of the C-terminal residues (266–323) are notshown in this figure.

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Thr-183 (a part of ar/R site), thereby occluding thechannel. These conformations of ar/R site argininehave also been revealed during MD simulations (Wang,Schulten, & Tajkhorshid, 2005). Similar interactionbetween Ala-181 and Arg-187 at ar/R site has also beennoticed in electron diffraction structure of AQP0 (Han,Guliaev, Walian, & Jap, 2006). Also, the file of waterwas broken near His-95 located on loop B in cytoplas-mic half of rAQP4 channel. This process of disruptionof water movement near His-95 occurred during all

simulations of rAQP4, irrespective of state of phosphory-lation and thus, could not be attributed to phosphoryla-tion of rAQP4. Further, similar event has beenpreviously reported during MD simulation of bovineAQP1 (Smolin, Li, Beck, & Daggett, 2008). Watermolecules were being constantly exchanged between thebulk water and those present near the cytoplasmic mouthduring simulations. The osmotic permeabilities (ρf) of5.98� 10�14 cm3s�1 and 6.24� 10�14 cm3s�1 wereobserved for phosphorylated and unphosphorylated

Figure 7. (A) C-terminal region (orange in color) of monomer A appear to cover the mouth of water channel of rAQP4-Ip.However, a gap between C-terminal region and mouth of channel is evident in the side view (below) of monomer A. (B) Inmonomer B, C-terminal region (magenta) was not able to cover the mouth of water channel completely. Below is the side view ofthe same. C-terminal regions of monomers C are shown in green color while transmembrane helices are blue colored.

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rAQP4-I, respectively, while diffusive permeability con-stants (ρd) of 0.84� 10�14 cm3s�1 and 0.90� 10�14

cm3s�1 are observed for the respective rAQP4-I(s). ρf/ρdratios of 6.93 and 7.12 for unphosphorylated andphosphorylated rAQP4 are comparable those obtainedfor AQP4 and other aquaporins in study by Han et al.(2006) and Hashido, Kidera, and Ikeguchi (2007). Exper-imental values of the osmotic permeability ranging from3.5 to 9 have been reported for AQP4 by Jung et al.(1994). The amount of discrepancy between these experi-mental and calculated values has been satisfactory takingaccount large errors in the calculated values and widevariety of experimental data as proposed by Hashidoet al. (2007).

Effect of phosphorylation on flow of water acrossrAQP4

RMSD of pore-lining residues of unphosphorylatedrAQP4 was found to be a little above 2.2Å, whereas inphosphorylated model rAQP4-Ip, these residues showedslightly lower RMSD of 1.8 Å. This reflects a little sta-bility of interior residues of the phosphorylated model ofaquaporin channel. Comparison of simulations ofrAQP4-I and that of rAQP4-Ip showed that the phos-phorylation of Ser-180 did not have any effect on resi-dues lining the interior of channel. The diameter of ar/Rconstriction site was also found to be unaffected in caseof rAQP4-Ip. Thus, this ruled out the possibility ofaction at a distance mechanism that may have beenexpected due to phosphorylation of Ser-180. Mitsumaet al. (2010) have mutated Ser-180 with aspartic acid inrAQP4 to mimic phosphorylation and have not observedany significant difference in its structure from that ofwild type rAQP4. Even in case of spinach aquaporin,SoPIP2;1, no action at a distance mechanism due tophosphorylation has been observed (Nyblom et al., 2009;Tornroth-Horsefield et al., 2006).

The interaction of phosphoserine-180 was observedonly with two residues (i.e. Arg-260 and Lys-263) of C-terminal helix. However, side chain of Arg-260 (amongKRR) and Lys-263 of C-terminal helix did not lead toblocking of the cytoplasmic mouth after interacting withphosphoserine-180. In fact, this interaction further stabi-lizes the open conformation of loop D and thereby keepsit away from the mouth of the channel (Figure 6). Thus,C-terminal helix was observed to come closer to thecytoplasmic mouth of channel within 10–15 ns ofsimulation time due to the interaction between phospho-serine-180 and residues of C-terminal region in mono-mers B and D. Also, the orientation of C-terminal helixbecame nearly perpendicular to the channel axis inmonomers B and D (Figure 6).

Only two of the cytoplasmic loop residues i.e.Lys-181 and Lys-109 are involved in interactions withphosphoserine-180. The side chain of Lys-109 did not

get extended into the channel in order to interact withphosphoserine-180 as it lies in its close proximity andon the same side of the cytoplasmic mouth and hence,can not provide any hindrance to the channel mouth.The C-terminal region exhibited movement towards thecytoplasmic mouth as two residues i.e. His-300 andLys-311 in addition to amino acids in helical regionshowed interactions with phosphoserine-180 in mono-mers C and A, respectively. Also, the C-terminal helicesof monomers A and C were engaged in multiple interac-tions with the neighboring monomers. Despite the factthat C-terminal region came closer and over the topof cytoplasmic mouth in all the monomers duringsimulations of rAQP4-Ip, it was not able to cover thecytoplasmic mouth of the channel completely. There wasa clear gap between the cytoplasmic mouth and theC-terminal region, though the size of this gap variedamong different monomers. These gaps which are foundin all the monomers of both unphosphorylated and phos-phorylated rAQP4 are irregular, triangular, or roughlyrectangular in shape. The size of these gaps may varyfrom 32 to 38ÅA and from 25 to 32ÅA forphosphorylated and unphosphoryalted rAQP4 across allmonomers of rAQP4, respectively. Thus, these gaps inC-terminal region and cytoplasmic mouth of aquaporinswill allow the movement of water in phosphorylated aswell unphosphorylated rAQP4. In addition, the interac-tions between C-terminal regions and loop D of two ofthe neighboring monomers also appear to prevent thecomplete bending of C-terminus over the cytoplasmicmouth of the water channel. However, such interactionswere missing in other monomers. Further, the C-terminalregion was found to lie spreaded over cytoplasmic mouthin phosphorylated monomers (Figure 7) as compared tounphosphorylated ones. It may appear from figure thatC-terminal region covered the cytoplasmic mouth of therAQP4, but it is not the case as seen in the side view(Figure 7) of the same monomer. A continuous flow ofwater was observed during the simulations and there wasa little difference in the osmotic permeability ofwater across the channel in both phosphorylated andunphosphorylated rAQP4.

In one of the recent structural studies on SoPIP2;1,phosphorylation induced structural perturbation has beenshown to displace a blocking residue away from channelso that the water flux through channel continues(Tornroth-Horsefield et al., 2006). On similar thoughts, ifphosphorylation were to cause the opposite effect i.e.decrease in water flow in rAQP4, it would be expected toinsert a blocking residue into the water channel. However,this does not appear to happen in the present case. Oursimulations of rAQP4 did reveal covering of thecytoplasmic mouth of rAQP4 after close association ofphosphoserine-180 or cytoplasmic loops with residues ofC-terminal region and no phosphorylation-induced

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conformational change leading to blocking of water chan-nel. But, water flowed unrestricted across the channel evenafter phosphorylation of Ser-180. The present MDsimulations suggest marginal effect to support the phos-phorylation-induced regulation of rAQP4 through gating.The studies of Mitsuma et al. (2010) have also observedno effect on structure of rAQP4 upon mimicking the phos-phorylation by mutating Ser-180 with aspartic acid. How-ever, C-terminal region was missing in their study andtherefore, its role has not been taken into account. In thelight of above, there could be a mechanism other than gat-ing by which regulation of rAQP4 may take place. Thefindings of Moeller, Fenton, Zeuthen, and Macaulay(2009) wherein reduction in water permeability of rAQP4was attributed to internalization of rAQP4 rather than gat-ing, could be a possibility.

Conclusions

The structure of C-terminal residues (255–323) wasmodeled in order to understand the role of C-terminalregion on phosphorylation induced regulation of rAQP4via gating. MD studies were carried out to studystructural changes in the phosphorylated rAQP4 in com-parison with unphosphorylated. It was observed duringsimulations that positively charged residues like His-300,Lys-311, Lys-109, Arg-260, and Lys-263, etc. located onthe C-terminal region as well as on the cytoplasmicloops connecting transmembrane helices come close tophosphoserine-180. These interactions between phospho-serine-180 and residues (Arg-260 and Lys-263) of C-terminal helix took place at the same side of cytoplasmicmouth and helped to stabilize the conformation of loopD, thus keeping it away from entrance of channel. MDsimulation of unphosphorylated rAQP4 did revealassociation of C-terminus and cytoplasmic loops involv-ing residues other than Ser-180 but only in case ofmonomer C. Although C-terminal region moved towardscytoplasmic mouth of the rAQP4, but the gap formedbetween these was larger than that observed in case ofphosphorylated rAQP4. Hydrogen and ionic bondswere observed in the residues of three monomers ininter-monomer interactions involving their loop D andC-terminal regions. Though, interaction of phosphoser-ine-180 with residues of C-terminal region resulted inmovement of this region close to the cytoplasmic mouthbut it did not cover the mouth of the channel completely.MD simulations of phosphorylated rAQP4 did not revealany blocking residue that may get inserted into the chan-nel leading to its blockage upon phosphorylation. Therewas a continuous flow of water molecules from aqueousphase till cytoplasmic mouth in both unphosphorylatedand phosphorylated rAQP4. The osmotic permeability ofwater was a little less in phosphorylated rAQP4 ascompared to unphosphorylated one. The simulations

carried out in present study did not reveal gating effectson rAQP4 channel due to phosphorylation of Ser-180.

Supplementary material

The supplementary material for this paper is availableonline at http://dx.doi.10.1080/07391102.2013.780981.

Acknowledgments

Authors are thankful to the Council of Scientific and IndustrialResearch (CSIR), Government of India for financial assistance.RS is a recipient of Senior Research Fellowship granted byCSIR.

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