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Probing interactions between uranyl ions and lipid membrane by molecular dynamics simulation
Computational and Theoretical Chemistry 976 (2011) 130–134
Contents lists available at SciVerse ScienceDirect
Computational and Theoretical Chemistry journal homepage: www.elsevier.com/locate/comptc
Probing interactions between uranyl ions and lipid membrane by molecular dynamics simulation Ying-Wu Lin a,b,⇑, Li-Fu Liao a a b
School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, China
a r t i c l e
i n f o
Article history: Received 3 April 2011 Received in revised form 13 August 2011 Accepted 13 August 2011 Available online 31 August 2011 Keywords: Uranium Membrane POPE Modeling Fluidity
a b s t r a c t Uranium is harmful to human health due to its radioactivity and toxicity. The cellular penetration of uranyl ion (UO2þ 2 ) involves its interactions with membranes. Herein, we investigated the interactions between UO2þ 2 ions and a bilayer lipid membrane, composed of 1-palmitoyl-2-oleoyl-glycerophosphoethanolamine (POPE), using molecular dynamics simulation. By presenting an atomic view of interactions between UO2þ 2 and the head group of POPE, we further investigated the dynamic consequences of uranyl binding and revealed the immobilization effect on the lipid molecules. This study provides insights into the mechanism of UO2þ 2 toxicity towards membranes in biological systems. Crown Copyright Ó 2011 Published by Elsevier B.V. All rights reserved.
1. Introduction Uranium is one of the heaviest metals on Earth with long-lived radioactivity. Uranyl ion (UO2þ 2 ), the most stable form of uranium under physiological conditions, is highly toxic to human body [1]. Besides the radiation damage, it is due to the ability of UO2þ 2 to interact with both DNA [2,3] and proteins such as transferrin, ferritin and albumin [4–6], thereby disrupting the native function of these biomolecules. While it is significant to reveal the impacts of UO2þ on protein structure and function [7], UO2þ encounters 2 2 membranes first when it penetrates cells. It has been known for about thirty years that UO2þ tends to interact with membranes 2 in vivo, resulting in different membrane properties [8]. Meanwhile, due to the difficulty in obtaining good quantitative structure in the biologically relevant, fully hydrated, fluid phase by the intrinsic presence of fluctuations [9], detail interactions at an atomic level are still lacking for a better understanding of UO2þ impacts on 2 membranes. As a complementary approach, molecular dynamics (MD) simulations have been applied to gain atomic and time-dependent information that otherwise is difficult to obtain experimentally [10–13]. Lins and Straatsma performed a computer simulation of a bacterial lipopolysaccharide (LPS) membrane [14], and recently they showed that the uptake of UO2þ 2 ion by the membrane is an ⇑ Corresponding author at: School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China. Tel.: +86 734 8297922. E-mail address: [email protected] (Y.-W. Lin).
energetically favorable process relative to the uptake of other ions such as Na+, Ca2+ and Cl [15]. In addition, MD simulations can study other metal ions (Cd2+, Pb2+, etc.) adsorption to bacterial surfaces [16]. The detail obtained by computer simulation can even be a guide to the interpretation of experimental results [17,18]. To provide deep insight into interactions between UO2þ and 2 membranes at an atomic level, we herein selected a common lipid membrane model of 1-palmitoyl-2-oleoyl-glycerophosphoethanolamine (POPE) (Fig. 1A), and performed a 15 ns MD simulation of its interactions with five UO2þ 2 ions. Moreover, we investigated the dynamic consequences of uranyl binding to membrane by performing a simulation of a control system of pure POPE bilayers with the same hydration level, which has not been attempted yet by MD simulations. 2. Methods The initial conformation of POPE bilayers in a liquid phase was constructed using program VMD 1.9 (Visual Molecular Dynamics) [19], which contains 30 POPE residues on each layer with 10 Å TIP3 water in z direction, resulting in a 50 50 60 Å3 cubic box (Fig. 1B). Five UO2þ 2 ions were introduced into the +z water layer with 5 Å to the surface, and the uranyl–lipid system was then neutralized by adding 10 Cl ions with a final concentration of 0.05 M. Using program NAMD 2.7 (Nanoscale Molecular Dynamics) [20], the system was first minimized for 5000 steps (2 fs per step) with conjugate gradient method at 0 K, and subsequently equilibrated for 10,000 steps at 300 K, then further minimized for 5000 steps.
2210-271X/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.comptc.2011.08.016
Y.-W. Lin, L.-F. Liao / Computational and Theoretical Chemistry 976 (2011) 130–134
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Fig. 1. (A) Chemical structure of POPE; (B) Equilibrated POPE bilayer membrane in a periodic box with five UO2þ 2 ions (indicated by an arrow) interacting with POPE residues in the +z direction.
Fig. 2. Atomic interactions between UO2þ 2 and one (A), two (B), or three (C) POPE residues and water molecules, as well as a chloride ion (D).
The resultant system was then subjected to a MD simulation for 15 ns with periodic boundary conditions via an NPT ensemble, where the number of particles N, the pressure P, and the temperature T of the system are kept constant at 300 K. A control MD
simulation of POPE bilayers in the absence of uranyl ions was also performed under identical conditions. A classical force field of CHARMM27 [21] was used for the POPE lipid system. The parameters used for UO2þ were developed and validated in previous 2
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studies [15,22], with a bond length of U–O 1.77 Å and an angle of O–U–O 180°. Visualization and data analysis were performed using program VMD 1.9.
3. Results and discussion 3.1. Uranyl binding to POPE After energy minimization and equilibration (Fig. 1B), the UO2þ 2 –POPE system adopts a bilayer structure with the orientations of individual lipids in good agreement with the simulation carried out in a liquid phase [10]. With the help of visual program VMD, we can probe the interactions between UO2þ 2 and POPE at an atomic level. It was found that UO2þ 2 ions can bind to 1–3 POPE residues during simulation, where one of two oxygen atoms of phosphoryl group in POPE coordinates to the uranium atom, with 5–3 water molecules making up the six-coordination, as shown in Fig. 2A– C. In addition, the counter ion, chloride, as used in MD simulation, was also found to be capable of binding to UO2þ (Fig. 2D). This 2 observation agrees with a recent study on uranyl chloride complexes in solution [23]. In chemistry, the chloride ions also form coordinating complex with UO2þ 2 , although the complex is much weaker compared to that of other inorganic ligands such as phosphate and carbonate [24]. Note that when UO2þ 2 bound to the phosphoryl group, there is no exchange of coordinating ligands with water molecules, suggesting that uranyl binding is an energy favorable process [15]. The average area per POPE head group was determined to be 65 Å2 at the end of simulation, which is slightly smaller than that in control simulation of pure POPE bilayers in the absence of UO2þ 2 ions (70 Å2). Since the amount of intercalated water in the polar region correlates with the interfacial area per lipid and is modulated by hydration effects [25], uranyl binding likely decreases
the number of intercalated water, thereby decreasing the fluidity of the bilayer membrane (see further analysis in the next section). An analysis of radial distribution function (RDF) for UO2þ 2 –POPE interactions shows that the average distance is 2.64 Å for uranylphosphoryl coordination, and the average coordination number is 1.2 and 0.5 for the two phosphoryl oxygen atoms, respectively (Fig. 3A and B). Previous crystallography study showed that in complex with an asparagine synthetase B (PDB entry 1CT9), the phosphoryl group of adenosine monophosphate (AMP) coordinates to two UO2þ 2 ions with a distance of 2.65 Å and 2.46 Å, respectively [26]. Note that there is no distance reported for the uranylphosphoryl coordination in the previous simulation study [15]. The analysis of RDF for uranyl–water interactions shows that the average coordination number of water is about five with an average distance of 2.78 Å to uranium atom (Fig. 3C). The second solvation shell peak is found to be around 5.0 Å, close to previous calculations (4.80 Å [15] and 4.70 Å [27]). In a previous study [15], Lins et al. showed that the coordination of five water molecules to uranyl was the dominating configurations during a 5 ns simulation, and the RDF for the uranium with respect to the water oxygen atoms presents a major peak at about 2.50 Å. This value is slightly shorter than our present result (2.78 Å), likely due to two different force fields employed for the lipid system. Meanwhile, although two simulations do not agree well with each other, both our and previous results are within the range of distances (2.64 ± 0.33 Å) observed for water coordinating to uranium in well-solved protein crystal structures [7]. Furthermore, the RDF for the uranium with respect to the chloride ion presents a major peak at 3.03 Å (Fig. 3D), indicating a weak interaction between Cl and UO2þ 2 . At the same time, the average coordination number is found to be as low as 0.03 (Fig. 3D, inset), suggesting that Cl has little chance to interact with UO2þ 2 in present MD simulation with a concentration of Cl 0.05 M. Note that the distribution of Cl ions in the water layer was generated
Fig. 3. Radial distribution function and average coordination numbers (inset) for uranyl-phosphoryl (A and B), uranyl–water (C), and uranyl-chloride (D) in the 15 ns simulation process.
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randomly by using the autoionize plug-in of program VMD 1.9 [20]. On the other hand, a resent study shows that for the uranyl chloride complexes in solution, the average coordination number of Cl increases as a function of chloride concentration, i.e. from 0.35 at 0.5 M to 2.3 at 4.8 M) [23].
3.2. Effects of uranyl binding to POPE In present simulation, we introduced UO2þ 2 ions only in the +z direction by mimicking the uranyl uptake on the extracellular side of a cell membrane. The difference between the two lipid layers lies thus only in uranyl binding. On the other hand, uranyl binding may induce changes in structure and dynamics of the UO2þ free 2 leaflet. Therefore, in order to probe the dynamics consequences as a result of uranyl binding, we performed a control simulation of pure POPE bilayers and compared the results with that of UO2þ 2 –POPE simulation. We first monitored the backbone root-mean-square deviation (RMSD) for the POPE residues in two lipid layers in both UO2þ 2 – POPE and pure POPE systems. As shown in Fig. 4, POPE residues 1–30 in the +z direction with UO2þ 2 bound to some residues exhibit an average RMSD value around 4 Å after 4 ns, which is slightly lower compared to that of POPE residues 31–60 in the z direction (around 5.5 Å). This observation suggests that uranyl binding leads to a conformational stabilization of POPE by direct coordination to the phosphoryl group(s) from one to three POPE residues (Fig. 2A– C). On the other hand, in the absence of UO2þ 2 ions, control simulation shows that POPE residues in both layers have an average RMSD value around 7 Å after 2 ns, close to a recent MD simulation study of POPE performed at 300 K using a MMFF94x force field (8.46 ± 1.06 Å) [28]. This observation suggests that uranyl binding has a long range stabilization effect on POPE in the z direction through hydrophobic interactions as well as van der Waals interactions between the tail groups of POPE in two monolayers. The order parameters (SCD) quantify the relative order of the hydrophobic lipid tails and provide structural and dynamical information on lipids [10,11]. In order to probe the conformational consequences of uranyl binding to POPE, we determined SCD of both saturated (sn-1) and unsaturated (sn-2) tails of POPE in UO2þ – 2 POPE and control POPE systems. As shown in Fig. 5, both profiles show a plateau in carbons 3–13 for POPE-sn-1 and a characteristic dip in double bond of C9-C10 for POPE-sn-2, which agrees well with previous simulation study [10,11] as well as experimental observations on lipids [29,30]. The dip in the SCD profile of POPE-sn-2 is not due to increased fluctuations but that it is rather a pure geometric effect [10]. In control simulation of pure POPE without UO2þ ions, the plateau of POPE-sn-1 is found to be 2 0.18, which agrees well with a very recent study using NMR experiments (0.18) and MD simulations (0.2) [31]. With UO2þ 2
Fig. 4. Rsidue backbone RMSD as a function of time for POPE residues (1–30) in +z direction with UO2þ ions bound and POPE resides (30–60) in z direction in the 2 absence of UO2þ 2 ions. The plots of POPE resides in bilayers in control simulation are shown for comparison.
Fig. 5. The lipid order parameters (SCD) of the two tail groups (sn-1, saturated chain, and sn-2, unsaturated chain) of POPE as determined from simulation of UO2þ 2 –POPE and control POPE systems, respectively.
bound, POPE-sn-1 shows a higher plateau at 0.22, and the same is true for the other tail, POPE-sn-2, suggesting that POPE tails are more ordered in presence of UO2þ 2 ions than that in pure POPE system as a result of uranyl binding to the head phosphoryl group of POPE. Moreover, we analyzed the dihedral of C7–C8–C9–C10 associated with the double bond of C9–C10 in the unsaturated tail of POPE during MD simulations, of which POPE-11 residue is shown as an example (Fig. 6). Although the double bond adopts a cis-conformation in the entire simulation process as observed previously [10], it was found that with UO2þ 2 bound, POPE residue has a lower positive–negative change frequency for the dihedral (1.5–2.5 ns) (Fig. 6A), as compared to that of metal-free form of POPE in control simulation (0.5–1.5 ns) (Fig. 6B). This observation further suggests that uranyl binding has an immobilization effect on POPE, which in turn, may reduce the fluidity of the bilayer lipid membrane. Additionally, to further probe the dynamics properties of POPE upon uranyl binding, we monitored the average angle between the two tail groups, sn-1 and sn-2, (\C16–C2–C18, Fig. 1A) of POPE residues coordinated to UO2þ ions during the 15 ns simulation 2
Fig. 6. The dihedral of C7–C8–C9–C10 in the tail group sn-2 of POPE-11 as a function of time for UO2þ 2 –POPE simulation (A) and control simulation (B). The conformation of the tail group with a positive and a negative dihedral of C7–C8–C9– C10 are shown respectively, as indicated by dashed arrows.
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developed by the Theoretical Biophysics Group in the Beckman Institute for Advanced Science and Technology at the University of Illinois at Urbana-Champaign, USA. This work was supported by the National Natural Science Foundation of China, NSFC (Nos. 21101091, 10975069) and Hunan Provincial Natural Science Foundation of China (No. 11JJ4017). References
Fig. 7. The average angles between the tail groups of POPE coordinated to UO2þ 2 as a function of time. The plot of the corresponding POPE residues in control simulation is shown for comparison. View of the representative shapes of POPE in the initial and final stages of two simulations, with dash line indicating the angle (inset).
process. The average angle for the corresponding POPE residues in control simulation was also monitored for comparison. As shown in Fig. 7, both POPE and UO2þ 2 –POPE have an initial average angle of 15°, and the angle increases to around 32° for POPE after 2 ns and around 25° for UO2þ 2 –POPE after 4 ns, respectively. Four representative shapes of POPE in the initial and final stages of two simulations are also shown in Fig. 7, inset. These observations indicate that uranyl binding to the head group of POPE results in a lower mobility of the tail groups, in agreement with the RMSD and order parameters analysis. Since the dynamics property of lipid membrane is close linked to its functions in metal ions uptake as well as protein translocation across the membrane [32,33], the UO2þ 2 ions binding may disrupt the cellular uptake of essential metal ions and even block protein entering in or releasing from cells by the low fluidity of lipids, thereby causing a high toxicity in biological systems. 4. Conclusions In summary, we probed the interactions between several UO2þ 2 ions and a common POPE lipid membrane by MD simulations, and 2þ presented an atomic view of UO2 ions coordinating to the phosphoryl group in POPE residue as well as surrounding water molecules or even chloride ion. Moreover, we investigated the dynamics consequences of uranyl binding and found that it immobilizes the lipid residue to some extent, as compared with control simulation. The information gained in this study sheds light on the mechanisms of uranyl toxicity towards membranes. Further investigation of uranyl impacts on membrane–protein system, such as UO2þ 2 interacting with a membrane protein, cytochrome b5, by both experimental and theoretical methods [34], is currently ongoing. Acknowledgments We thank Dr. Tianlei Ying at the National Institutes of Health (NIH), USA, for helpful discussions. NAMD and VMD were
[1] E.S. Craft, A.W. Abu-Qare, M.M. Flaherty, M.C. Garofolo, H.L. Rincavage, M.B. Abou-Donia, J. Toxicol. Environ. Health B 7 (2004) 297. [2] J.H. Lee, Z. Wang, Y. Lu, J. Am. Chem. Soc. 130 (2008) 14217. [3] A.K. Brown, J. Liu, Y. He, Y. Lu, Chem. Biochem. 10 (2009) 486. [4] C. Vidaud, S. Gourion-Arsiquaud, F. Rollin-Genetet, C. Torne-Celer, S. Plantevin, O. Pible, C. Berthomieu, E. Quéméneur, Biochemistry 46 (2007) 2215. [5] G. Montavon, C. Apostolidis, F. Bruchertseifer, U. Repinc, A. Morgenstern, J. Inorg. Biochem. 103 (2009) 1609. [6] J. Michon, S. Frelon, C. Garnier, F. Coppin, J. Fluoresc. 20 (2010) 581. [7] O. Pible, P. Guilbaud, J.-L. Pellequer, C. Vidaud, E. Quéméneur, Biochimie 88 (2006) 1631. [8] H.P. Ting-Beall, J. Microsc. 118 (1980) 221. [9] J.F. Nagle, S. Tristram-Nagle, Biochim. Biophys. Acta 1469 (2000) 159. [10] H. Heller, M. Schaefer, K. Schulten, J. Phys. Chem. 97 (1993) 8343. [11] D. Lupyan, M. Mezei, D.E. Logothetis, R. Osman, Biophys. J. 98 (2010) 240. [12] F. Sepulcre, A. Cordomí, M.G. Proietti, J.J. Perez, J. García, E. Querol, E. Padrós, Proteins 67 (2007) 360. [13] Y.W. Lin, Proteins 79 (2011) 679. [14] R.D. Lins, T.P. Straatsma, Biophys. J. 81 (2001) 1037. [15] R.D. Lins, E.R. Vorpagel, M. Guglielmi, T.P. Straatsma, Biomacromolecules 9 (2008) 29. [16] K.J. Johnson, R.T. Cygan, J.B. Fein, Geochim. Cosmochim. Acta 70 (2006) 5075. [17] H.I. Petrache, K. Tu, J.F. Nagle, Biophys. J. 76 (1999) 2479. [18] J.F. Nagle, R. Zhang, S. Tristram-Nagle, W. Sun, H.I. Petrache, R.M. Suter, Biophys. J. 70 (1996) 1419. [19] W. Humphrey, A. Dalke, K. Schulten, J. Mol. Graph. 14 (1996) 33. [20] L. Kalé, R. Skeel, M. Bhandarkar, R. Brunner, A. Gursoy, N. Krawetz, J. Phillips, A. Shinozaki, K. Varadarajan, K. Schulten, J. Comput. Phys. 151 (1999) 283. [21] A.D. MacKerell Jr., D. Bashford, M. Bellott, R.L. Dunbrack Jr., J. Evanseck, M.J. Field, S. Fischer, J. Gao, H. Guo, S. Ha, D. Joseph, L. Kuchnir, K. Kuczera, F.T.K. Lau, C. Mattos, S. Michnick, T. Ngo, D.T. Nguyen, B. Prodhom, I.W.E. Reiher, B. Roux, M. Schlenkrich, J. Smith, R. Stote, J. Straub, M. Watanabe, J. WiorkiewiczKuczera, D. Yin, M. Karplus, J. Phys. Chem. B 102 (1998) 3586. [22] P. Guilbaud, G. Wipff, J. Mol. Strut. Theochem 366 (1996) 55. [23] L. Soderholm, S. Skanthakumar, R.E. Wilson, J. Phys. Chem. A. 115 (2011) 4959. [24] R. Guillaumont, T. Fanghanel, J. Fuger, I. Grenthe, V. Neck, D.A. Palmer, M.H. Rand, Update on the Chemical Thermodynamics of Uranium, Neptunium, Plutonium, Americium, and Technetium, vol. 5, Elsevier, Amsterdam, 2003 (pp. 970). [25] E.A. Disalvo, F. Lairion, F. Martini, E. Tymczyszyn, M. Frías, H. Almaleck, G.J. Gordillo, Biochim. Biophys. Acta 1778 (2008) 2655. [26] T.M. Larsen, S.K. Boehlein, S.M. Schuster, N.G. Richards, J.B. Thoden, H.M. Holden, I. Rayment, Biochemistry 38 (1999) 16146. [27] D. Hagberg, G. Karlström, B.O. Roos, L. Gagliardi, J. Am. Chem. Soc. 127 (2005) 14250. [28] S. Shityakov, T. Dandekar, Nano 6 (2011) 19. [29] A. Seelig, J. Seelig, Biochemistry 16 (1977) 45. [30] M. Lafleur, P.R. Cullis, M. Bloom, Eur. Biophys. J. 19 (1990) 55. [31] T.D. Romo, L.A. Bradney, D.V. Greathouse, A. Grossfield, Biochim. Biophys. Acta 1808 (2011) 2019. [32] M. Rahman, S.G. Patching, F. Ismat, P.J. Henderson, R.B. Herbert, S.A. Baldwin, M.J. Mc Pherson, Mol. Membr. Biol. 25 (2008) 683. [33] W. Wickner, R. Schekman, Science 310 (2005) 1452. [34] D. Wan, L.F. Liao, M.M. Zhao, M.L. Wu, Y.M. Wu, Y.W. Lin, J. Mol. Model. (2011), doi:10.1007/s00894-011-1097-1.
Contents lists available at SciVerse ScienceDirect
Computational and Theoretical Chemistry journal homepage: www.elsevier.com/locate/comptc
Probing interactions between uranyl ions and lipid membrane by molecular dynamics simulation Ying-Wu Lin a,b,⇑, Li-Fu Liao a a b
School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210093, China
a r t i c l e
i n f o
Article history: Received 3 April 2011 Received in revised form 13 August 2011 Accepted 13 August 2011 Available online 31 August 2011 Keywords: Uranium Membrane POPE Modeling Fluidity
a b s t r a c t Uranium is harmful to human health due to its radioactivity and toxicity. The cellular penetration of uranyl ion (UO2þ 2 ) involves its interactions with membranes. Herein, we investigated the interactions between UO2þ 2 ions and a bilayer lipid membrane, composed of 1-palmitoyl-2-oleoyl-glycerophosphoethanolamine (POPE), using molecular dynamics simulation. By presenting an atomic view of interactions between UO2þ 2 and the head group of POPE, we further investigated the dynamic consequences of uranyl binding and revealed the immobilization effect on the lipid molecules. This study provides insights into the mechanism of UO2þ 2 toxicity towards membranes in biological systems. Crown Copyright Ó 2011 Published by Elsevier B.V. All rights reserved.
1. Introduction Uranium is one of the heaviest metals on Earth with long-lived radioactivity. Uranyl ion (UO2þ 2 ), the most stable form of uranium under physiological conditions, is highly toxic to human body [1]. Besides the radiation damage, it is due to the ability of UO2þ 2 to interact with both DNA [2,3] and proteins such as transferrin, ferritin and albumin [4–6], thereby disrupting the native function of these biomolecules. While it is significant to reveal the impacts of UO2þ on protein structure and function [7], UO2þ encounters 2 2 membranes first when it penetrates cells. It has been known for about thirty years that UO2þ tends to interact with membranes 2 in vivo, resulting in different membrane properties [8]. Meanwhile, due to the difficulty in obtaining good quantitative structure in the biologically relevant, fully hydrated, fluid phase by the intrinsic presence of fluctuations [9], detail interactions at an atomic level are still lacking for a better understanding of UO2þ impacts on 2 membranes. As a complementary approach, molecular dynamics (MD) simulations have been applied to gain atomic and time-dependent information that otherwise is difficult to obtain experimentally [10–13]. Lins and Straatsma performed a computer simulation of a bacterial lipopolysaccharide (LPS) membrane [14], and recently they showed that the uptake of UO2þ 2 ion by the membrane is an ⇑ Corresponding author at: School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China. Tel.: +86 734 8297922. E-mail address: [email protected] (Y.-W. Lin).
energetically favorable process relative to the uptake of other ions such as Na+, Ca2+ and Cl [15]. In addition, MD simulations can study other metal ions (Cd2+, Pb2+, etc.) adsorption to bacterial surfaces [16]. The detail obtained by computer simulation can even be a guide to the interpretation of experimental results [17,18]. To provide deep insight into interactions between UO2þ and 2 membranes at an atomic level, we herein selected a common lipid membrane model of 1-palmitoyl-2-oleoyl-glycerophosphoethanolamine (POPE) (Fig. 1A), and performed a 15 ns MD simulation of its interactions with five UO2þ 2 ions. Moreover, we investigated the dynamic consequences of uranyl binding to membrane by performing a simulation of a control system of pure POPE bilayers with the same hydration level, which has not been attempted yet by MD simulations. 2. Methods The initial conformation of POPE bilayers in a liquid phase was constructed using program VMD 1.9 (Visual Molecular Dynamics) [19], which contains 30 POPE residues on each layer with 10 Å TIP3 water in z direction, resulting in a 50 50 60 Å3 cubic box (Fig. 1B). Five UO2þ 2 ions were introduced into the +z water layer with 5 Å to the surface, and the uranyl–lipid system was then neutralized by adding 10 Cl ions with a final concentration of 0.05 M. Using program NAMD 2.7 (Nanoscale Molecular Dynamics) [20], the system was first minimized for 5000 steps (2 fs per step) with conjugate gradient method at 0 K, and subsequently equilibrated for 10,000 steps at 300 K, then further minimized for 5000 steps.
2210-271X/$ - see front matter Crown Copyright Ó 2011 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.comptc.2011.08.016
Y.-W. Lin, L.-F. Liao / Computational and Theoretical Chemistry 976 (2011) 130–134
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Fig. 1. (A) Chemical structure of POPE; (B) Equilibrated POPE bilayer membrane in a periodic box with five UO2þ 2 ions (indicated by an arrow) interacting with POPE residues in the +z direction.
Fig. 2. Atomic interactions between UO2þ 2 and one (A), two (B), or three (C) POPE residues and water molecules, as well as a chloride ion (D).
The resultant system was then subjected to a MD simulation for 15 ns with periodic boundary conditions via an NPT ensemble, where the number of particles N, the pressure P, and the temperature T of the system are kept constant at 300 K. A control MD
simulation of POPE bilayers in the absence of uranyl ions was also performed under identical conditions. A classical force field of CHARMM27 [21] was used for the POPE lipid system. The parameters used for UO2þ were developed and validated in previous 2
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studies [15,22], with a bond length of U–O 1.77 Å and an angle of O–U–O 180°. Visualization and data analysis were performed using program VMD 1.9.
3. Results and discussion 3.1. Uranyl binding to POPE After energy minimization and equilibration (Fig. 1B), the UO2þ 2 –POPE system adopts a bilayer structure with the orientations of individual lipids in good agreement with the simulation carried out in a liquid phase [10]. With the help of visual program VMD, we can probe the interactions between UO2þ 2 and POPE at an atomic level. It was found that UO2þ 2 ions can bind to 1–3 POPE residues during simulation, where one of two oxygen atoms of phosphoryl group in POPE coordinates to the uranium atom, with 5–3 water molecules making up the six-coordination, as shown in Fig. 2A– C. In addition, the counter ion, chloride, as used in MD simulation, was also found to be capable of binding to UO2þ (Fig. 2D). This 2 observation agrees with a recent study on uranyl chloride complexes in solution [23]. In chemistry, the chloride ions also form coordinating complex with UO2þ 2 , although the complex is much weaker compared to that of other inorganic ligands such as phosphate and carbonate [24]. Note that when UO2þ 2 bound to the phosphoryl group, there is no exchange of coordinating ligands with water molecules, suggesting that uranyl binding is an energy favorable process [15]. The average area per POPE head group was determined to be 65 Å2 at the end of simulation, which is slightly smaller than that in control simulation of pure POPE bilayers in the absence of UO2þ 2 ions (70 Å2). Since the amount of intercalated water in the polar region correlates with the interfacial area per lipid and is modulated by hydration effects [25], uranyl binding likely decreases
the number of intercalated water, thereby decreasing the fluidity of the bilayer membrane (see further analysis in the next section). An analysis of radial distribution function (RDF) for UO2þ 2 –POPE interactions shows that the average distance is 2.64 Å for uranylphosphoryl coordination, and the average coordination number is 1.2 and 0.5 for the two phosphoryl oxygen atoms, respectively (Fig. 3A and B). Previous crystallography study showed that in complex with an asparagine synthetase B (PDB entry 1CT9), the phosphoryl group of adenosine monophosphate (AMP) coordinates to two UO2þ 2 ions with a distance of 2.65 Å and 2.46 Å, respectively [26]. Note that there is no distance reported for the uranylphosphoryl coordination in the previous simulation study [15]. The analysis of RDF for uranyl–water interactions shows that the average coordination number of water is about five with an average distance of 2.78 Å to uranium atom (Fig. 3C). The second solvation shell peak is found to be around 5.0 Å, close to previous calculations (4.80 Å [15] and 4.70 Å [27]). In a previous study [15], Lins et al. showed that the coordination of five water molecules to uranyl was the dominating configurations during a 5 ns simulation, and the RDF for the uranium with respect to the water oxygen atoms presents a major peak at about 2.50 Å. This value is slightly shorter than our present result (2.78 Å), likely due to two different force fields employed for the lipid system. Meanwhile, although two simulations do not agree well with each other, both our and previous results are within the range of distances (2.64 ± 0.33 Å) observed for water coordinating to uranium in well-solved protein crystal structures [7]. Furthermore, the RDF for the uranium with respect to the chloride ion presents a major peak at 3.03 Å (Fig. 3D), indicating a weak interaction between Cl and UO2þ 2 . At the same time, the average coordination number is found to be as low as 0.03 (Fig. 3D, inset), suggesting that Cl has little chance to interact with UO2þ 2 in present MD simulation with a concentration of Cl 0.05 M. Note that the distribution of Cl ions in the water layer was generated
Fig. 3. Radial distribution function and average coordination numbers (inset) for uranyl-phosphoryl (A and B), uranyl–water (C), and uranyl-chloride (D) in the 15 ns simulation process.
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randomly by using the autoionize plug-in of program VMD 1.9 [20]. On the other hand, a resent study shows that for the uranyl chloride complexes in solution, the average coordination number of Cl increases as a function of chloride concentration, i.e. from 0.35 at 0.5 M to 2.3 at 4.8 M) [23].
3.2. Effects of uranyl binding to POPE In present simulation, we introduced UO2þ 2 ions only in the +z direction by mimicking the uranyl uptake on the extracellular side of a cell membrane. The difference between the two lipid layers lies thus only in uranyl binding. On the other hand, uranyl binding may induce changes in structure and dynamics of the UO2þ free 2 leaflet. Therefore, in order to probe the dynamics consequences as a result of uranyl binding, we performed a control simulation of pure POPE bilayers and compared the results with that of UO2þ 2 –POPE simulation. We first monitored the backbone root-mean-square deviation (RMSD) for the POPE residues in two lipid layers in both UO2þ 2 – POPE and pure POPE systems. As shown in Fig. 4, POPE residues 1–30 in the +z direction with UO2þ 2 bound to some residues exhibit an average RMSD value around 4 Å after 4 ns, which is slightly lower compared to that of POPE residues 31–60 in the z direction (around 5.5 Å). This observation suggests that uranyl binding leads to a conformational stabilization of POPE by direct coordination to the phosphoryl group(s) from one to three POPE residues (Fig. 2A– C). On the other hand, in the absence of UO2þ 2 ions, control simulation shows that POPE residues in both layers have an average RMSD value around 7 Å after 2 ns, close to a recent MD simulation study of POPE performed at 300 K using a MMFF94x force field (8.46 ± 1.06 Å) [28]. This observation suggests that uranyl binding has a long range stabilization effect on POPE in the z direction through hydrophobic interactions as well as van der Waals interactions between the tail groups of POPE in two monolayers. The order parameters (SCD) quantify the relative order of the hydrophobic lipid tails and provide structural and dynamical information on lipids [10,11]. In order to probe the conformational consequences of uranyl binding to POPE, we determined SCD of both saturated (sn-1) and unsaturated (sn-2) tails of POPE in UO2þ – 2 POPE and control POPE systems. As shown in Fig. 5, both profiles show a plateau in carbons 3–13 for POPE-sn-1 and a characteristic dip in double bond of C9-C10 for POPE-sn-2, which agrees well with previous simulation study [10,11] as well as experimental observations on lipids [29,30]. The dip in the SCD profile of POPE-sn-2 is not due to increased fluctuations but that it is rather a pure geometric effect [10]. In control simulation of pure POPE without UO2þ ions, the plateau of POPE-sn-1 is found to be 2 0.18, which agrees well with a very recent study using NMR experiments (0.18) and MD simulations (0.2) [31]. With UO2þ 2
Fig. 4. Rsidue backbone RMSD as a function of time for POPE residues (1–30) in +z direction with UO2þ ions bound and POPE resides (30–60) in z direction in the 2 absence of UO2þ 2 ions. The plots of POPE resides in bilayers in control simulation are shown for comparison.
Fig. 5. The lipid order parameters (SCD) of the two tail groups (sn-1, saturated chain, and sn-2, unsaturated chain) of POPE as determined from simulation of UO2þ 2 –POPE and control POPE systems, respectively.
bound, POPE-sn-1 shows a higher plateau at 0.22, and the same is true for the other tail, POPE-sn-2, suggesting that POPE tails are more ordered in presence of UO2þ 2 ions than that in pure POPE system as a result of uranyl binding to the head phosphoryl group of POPE. Moreover, we analyzed the dihedral of C7–C8–C9–C10 associated with the double bond of C9–C10 in the unsaturated tail of POPE during MD simulations, of which POPE-11 residue is shown as an example (Fig. 6). Although the double bond adopts a cis-conformation in the entire simulation process as observed previously [10], it was found that with UO2þ 2 bound, POPE residue has a lower positive–negative change frequency for the dihedral (1.5–2.5 ns) (Fig. 6A), as compared to that of metal-free form of POPE in control simulation (0.5–1.5 ns) (Fig. 6B). This observation further suggests that uranyl binding has an immobilization effect on POPE, which in turn, may reduce the fluidity of the bilayer lipid membrane. Additionally, to further probe the dynamics properties of POPE upon uranyl binding, we monitored the average angle between the two tail groups, sn-1 and sn-2, (\C16–C2–C18, Fig. 1A) of POPE residues coordinated to UO2þ ions during the 15 ns simulation 2
Fig. 6. The dihedral of C7–C8–C9–C10 in the tail group sn-2 of POPE-11 as a function of time for UO2þ 2 –POPE simulation (A) and control simulation (B). The conformation of the tail group with a positive and a negative dihedral of C7–C8–C9– C10 are shown respectively, as indicated by dashed arrows.
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developed by the Theoretical Biophysics Group in the Beckman Institute for Advanced Science and Technology at the University of Illinois at Urbana-Champaign, USA. This work was supported by the National Natural Science Foundation of China, NSFC (Nos. 21101091, 10975069) and Hunan Provincial Natural Science Foundation of China (No. 11JJ4017). References
Fig. 7. The average angles between the tail groups of POPE coordinated to UO2þ 2 as a function of time. The plot of the corresponding POPE residues in control simulation is shown for comparison. View of the representative shapes of POPE in the initial and final stages of two simulations, with dash line indicating the angle (inset).
process. The average angle for the corresponding POPE residues in control simulation was also monitored for comparison. As shown in Fig. 7, both POPE and UO2þ 2 –POPE have an initial average angle of 15°, and the angle increases to around 32° for POPE after 2 ns and around 25° for UO2þ 2 –POPE after 4 ns, respectively. Four representative shapes of POPE in the initial and final stages of two simulations are also shown in Fig. 7, inset. These observations indicate that uranyl binding to the head group of POPE results in a lower mobility of the tail groups, in agreement with the RMSD and order parameters analysis. Since the dynamics property of lipid membrane is close linked to its functions in metal ions uptake as well as protein translocation across the membrane [32,33], the UO2þ 2 ions binding may disrupt the cellular uptake of essential metal ions and even block protein entering in or releasing from cells by the low fluidity of lipids, thereby causing a high toxicity in biological systems. 4. Conclusions In summary, we probed the interactions between several UO2þ 2 ions and a common POPE lipid membrane by MD simulations, and 2þ presented an atomic view of UO2 ions coordinating to the phosphoryl group in POPE residue as well as surrounding water molecules or even chloride ion. Moreover, we investigated the dynamics consequences of uranyl binding and found that it immobilizes the lipid residue to some extent, as compared with control simulation. The information gained in this study sheds light on the mechanisms of uranyl toxicity towards membranes. Further investigation of uranyl impacts on membrane–protein system, such as UO2þ 2 interacting with a membrane protein, cytochrome b5, by both experimental and theoretical methods [34], is currently ongoing. Acknowledgments We thank Dr. Tianlei Ying at the National Institutes of Health (NIH), USA, for helpful discussions. NAMD and VMD were
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