Journal of Molecular Modeling

, Volume 16, Issue 10, pp 1625–1637 | Cite as

Contribution of charged and polar residues for the formation of the E1–E2 heterodimer from Hepatitis C Virus

  • Siti Azma Jusoh
  • Christoph Welsch
  • Shirley W. I. Siu
  • Rainer A. Böckmann
  • Volkhard HelmsEmail author
Original Paper


The transmembrane domains of the envelope glycoprotein E1 and E2 have crucial multifunctional roles in the biogenesis of hepatitis C virus. We have performed molecular dynamics simulations to investigate a structural model of the transmembrane segments of the E1–E2 heterodimer. The simulations support the key role of the Lys370–Asp728 ion pair for mediating the E1–E2 heterodimerization. In comparison to these two residues, the simulation results also reveal the differential effect of the conserved Arg730 residue that has been observed in experimental studies. Furthermore, we discovered the formation of inter-helical hydrogen bonds via Asn367 that stabilize dimer formation. Simulations of single and double mutants further demonstrate the importance of the ion-pair and polar interactions between the interacting helix monomers. The conformation of the E1 fragment in the simulation of the E1–E2 heterodimer is in close agreement with an NMR structure of the E1 transmembrane segment. The proposed model of the E1–E2 heterodimer supports the postulated cooperative insertion of both helices by the translocon complex into the bilayer.


Charged amino acids Hepatitis E1-E2 envelope glycoproteins Molecular dynamics simulation Salt-bridges Transmembrane helix 



We are grateful for the financial support of the Universiti Teknologi MARA, Malaysia and by grant I 80469 by the Volkswagen foundation and for the research facilities from The Center for Bioinformatics Saarland University. We also thank the Deutsche Forschungsgemeinschaft Graduate School 1276/1 “Structure Formation and Transport in Complex Systems” for collaboration.

Supplementary material

894_2010_672_MOESM1_ESM.pdf (12 kb)
Table S1 Average H-bonds analyzed for the data between 80 and 100 ns of MD simulations of E1–E2 wild-types and mutants. (PDF 11 kb)
894_2010_672_MOESM2_ESM.pdf (22 kb)
Fig. S1 Results from secondary structure prediction programs. The consensus prediction is given at the bottom; the positions in the consensus sequence indicate that three or more methods gave the same results. Highlighted in blue in the consensus prediction are the charged residues Lys370, Asp728, and Arg730. The dotted lines show the segments which were used in the MD simulations. (PDF 22 kb)
894_2010_672_MOESM3_ESM.pdf (51 kb)
Fig. S2 Final snapshot after 100 ns of MD simulations of the H-segment monomers containing a charged residue in the middle of their TM domains: (a) H-segment with charged Lys370 and (b) H-segment with charged Asp728. Lipid tails and ions are not shown for clarity. (PDF 50 kb)
894_2010_672_MOESM4_ESM.pdf (137 kb)
Fig. S3 Root-mean-square deviations (RMSDs) of E1 and E2 TM domains of the E1–E2 wild-type and mutant heterodimers. (A) RMSDs of wild-type E1–E2 heterodimers versus the E1 and E2 simulations of isolated helices. (B) RMSDs of single mutants which contain a salt-bridge at the helix-helix packing interface and (C) RMSDs of double mutants. In (B) and (C), the E1–E2 wild-type 1 is shown for comparison. (PDF 137 kb)
894_2010_672_MOESM5_ESM.pdf (305 kb)
Fig. S4 Final snapshots after 100 ns MD simulation of the E1–E2 heterodimers in the two wild-type simulations. The conserved residues Asn367, Lys370, Asp728, and Arg730 are highlighted as stick presentation. Lipid tails and ions are not shown for clarity. (PDF 305 kb)
894_2010_672_MOESM6_ESM.pdf (43 kb)
Fig. S5 Final snapshot of mutants (a) K370A and (b) D728A. The conserved residues Asn367, Lys370, Asp728, and Arg730 are shown as stick representation. The mutated residues, lipid bilayer, water and ions molecules are not shown for clarity. (PDF 43 kb)


  1. 1.
    Appel N, Schaller T, Penin F, Bartenschlager R (2006) From structure to function: New insights into hepatitis C virus RNA replication. J Biol Chem 281:9833–9836. doi: 10.1074/jbc.R500026200 CrossRefGoogle Scholar
  2. 2.
    Moradpour D, Penin F, Rice CM (2007) Replication of hepatitis C virus. Nat Rev Microbiol 5:453–463. doi: 10.1038/nrmicro1645 CrossRefGoogle Scholar
  3. 3.
    Bartosch B, Dubuisson J, Cosset F (2003) Infectious hepatitis C virus pseudo-particles containing functional E1–E2 envelope protein complexes. J Exp Med 197:633–642. doi: 10.1084/jem.20021756 CrossRefGoogle Scholar
  4. 4.
    Wakita T et al. (2005) Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11:905–905. doi: 10.1038/nm1268 CrossRefGoogle Scholar
  5. 5.
    Francki R, Fauquet C, Knudson D, Brown F (1991) Classification and nomenclature of viruses. Fifth report of the international Committee on taxonomy of viruses. Arch Virol Suppl 2:140–144Google Scholar
  6. 6.
    Lindenbach BD, Thiel HJ, Rice CM (2001) Flaviviridae: the viruses and their replication. In: Knipe DM, Howley PM (eds) Fields virology, 5th edn. Lippincott Williams & Wilkins, Philadelphia, pp 991–1041Google Scholar
  7. 7.
    Matsuura Y, Miyamura T (1993) The molecular biology of hepatitis C virus. Semin Virol 4:297–304. doi: 10.1006/smvy.1993.1026 CrossRefGoogle Scholar
  8. 8.
    Dubuisson J et al. (2000) Glycosylation of the hepatitis C virus envelope protein E1 is dependent on the presence of a downstream sequence on the viral polyprotein. J Biol Chem 275:30605–30609. doi: 10.1074/jbc.M004326200 CrossRefGoogle Scholar
  9. 9.
    Cocquerel L, Wychowski C, Minner F, Penin F, Dubuisson J (2000) Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a major role in the processing, subcellular localization, and assembly of these envelope proteins. J Virol 74:3623–3633CrossRefGoogle Scholar
  10. 10.
    Cuthbertson JM, Bond PJ, Sansom MSP (2006) Transmembrane helix-helix interactions: comparative simulations of the glycophorin A dimer. Biochemistry 45:14298–14310. doi: 10.1021/bi0610911 CrossRefGoogle Scholar
  11. 11.
    Ciczora Y, Callens N, Penin F, Pecheur EI, Dubuisson J (2007) Transmembrane domains of hepatitis C virus envelope glycoproteins: Residues involved in E1E2 heterodimerization and involvement of these domains in virus entry. J Virol 81:2372–2381. doi: 10.1128/JVI.02198-06 CrossRefGoogle Scholar
  12. 12.
    Cocquerel L et al. (2002) Topological changes in the transmembrane domains of hepatitis C virus envelope glycoproteins. EMBO J 21:2893–2902. doi: 10.1093/emboj/cdf295 CrossRefGoogle Scholar
  13. 13.
    Ciczora Y et al. (2005) Contribution of the charged residues of hepatitis C virus glycoprotein E2 transmembrane domain to the functions of the E1E2 heterodimer. J Gen Virol 86:2793–2798. doi: 10.1099/vir.0.81140-0 CrossRefGoogle Scholar
  14. 14.
    Ronecker S, Zimmer G, Herrler G, Greiser-Wilke I, Grummer B (2008) Formation of bovine viral diarrhea virus E1–E2 heterodimers is essential for virus entry and depends on charged residues in the transmembrane domains. J Gen Virol 89:2114–2121. doi: 10.1099/vir.0.2008/001792-0 CrossRefGoogle Scholar
  15. 15.
    White SH (2004) The progress of membrane protein structure determination. Protein Sci 13:1948. doi: 10.1110/ps.04712004 CrossRefGoogle Scholar
  16. 16.
    Bond PJ, Sansom MSP (2003) Membrane protein dynamics versus environment: simulations of OmpA in a micelle and in a bilayer. J Mol Biol 329:1035–1053. doi: 10.1016/S0022-2836(03)00408-X CrossRefGoogle Scholar
  17. 17.
    Böckmann RA, Caflisch A (2005) Spontaneous formation of detergent micelles around the outer membrane protein OmpX. Biophys J 88:3191–3204. doi: 10.1529/biophysj.105.060426 CrossRefGoogle Scholar
  18. 18.
    Lomize AL, Pogozheva ID, Lomize MA, Mosberg HI (2006) Positioning of proteins in membranes: a computational approach. Protein Sci 15:1318–1333. doi: 10.1110/ps.062126106 CrossRefGoogle Scholar
  19. 19.
    Matthews EE, Zoonens M, Engelman DM (2006) Dynamic helix interactions in transmembrane signaling. Cell 127:447–450. doi: 10.1016/j.cell.2006.10.016 CrossRefGoogle Scholar
  20. 20.
    Treutlein HR, Lemmon MA, Engelman DM, Brunger A (1992) The glycophorin A transmembrane domain dimer: sequence-specific propensity for a right-handed supercoil of helixes. Biochemistry 31:12726–12732. doi: 10.1021/bi00166a002 CrossRefGoogle Scholar
  21. 21.
    Lemmon MA, Flanagan JM, Treutlein HR, Zhang J, Engelman DM (1992) Sequence specificity in the dimerization of transmembrane. alpha.-helixes. Biochemistry 31:12719–12725. doi: 10.1021/bi00166a002 CrossRefGoogle Scholar
  22. 22.
    Adams PD, Engelman DM, Brünger AT (1996) Improved prediction for the structure of the dimeric transmembrane domain of glycophorin A obtained through global searching. Proteins 26:257–261. doi: 10.1002/(SICI)1097-0134(199611)26:3<257::AID-PROT2>3.0.CO;2-B CrossRefGoogle Scholar
  23. 23.
    MacKenzie KR, Prestegard JH, Engelman DM (1997) A transmembrane helix dimer: structure and implications. Science 276:131–133. doi: 10.1126/science.276.5309.131 CrossRefGoogle Scholar
  24. 24.
    Woolf TB (1998) Molecular dynamics simulations of individual alpha-helices of bacteriorhodopsin in dimyristoylphosphatidylcholine. II. Interaction energy analysis. Biophys J 74:115–131. doi: 10.1016/S0959-440X(99)80015-3 CrossRefGoogle Scholar
  25. 25.
    Candler A, Featherstone M, Ali R, Maloney L, Watts A, Fischer WB (2005) Computational analysis of mutations in the transmembrane region of Vpu from HIV-1. BBA-Biomembranes 1716:1–10. doi: 10.1016/j.bbamem.2005.07.012 CrossRefGoogle Scholar
  26. 26.
    Fischer WB, Sansom MSP (2002) Viral ion channels: structure and function. BBA-Biomembranes 1561:27–45. doi: 10.1016/S0304-4157(01)00009-0 CrossRefGoogle Scholar
  27. 27.
    Hénin J, Chipot C, Pohorille A (2005) Insights into the recognition and association of transmembrane α-helices. The free energy of α-helix dimerization in glycophorin A. J Am Chem Soc 127:8478–8484. doi: 10.1021/ja050581y CrossRefGoogle Scholar
  28. 28.
    Yoo J, Cui Q (2008) Does Arginine remain protonated in the lipid membrane? Insights from microscopic pKa calculations. Biophys J 94:61–63. doi: 10.1529/biophysj.107.122945 CrossRefGoogle Scholar
  29. 29.
    Wu CH et al. (2006) The Universal Protein Resource (UniProt): an expanding universe of protein information. Nucleic Acids Res 34:187–191. doi: 10.1093/nar/gkj161 CrossRefGoogle Scholar
  30. 30.
    Hessa T et al. (2005) Recognition of transmembrane helices by the endoplasmic reticulum translocon. Nature 433:377–381. doi: 10.1038/nature03216 CrossRefGoogle Scholar
  31. 31.
    Combet C et al. (2007) euHCVdb: the European hepatitis C virus database. Nucleic Acids Res 35:D363. doi: 10.1093/nar/gkl970 CrossRefGoogle Scholar
  32. 32.
    Simmonds P et al. (2005) Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 42:962–973. doi: 10.1002/hep.20819 CrossRefGoogle Scholar
  33. 33.
    Larkin MA et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947. doi: 10.1093/bioinformatics/btm404 CrossRefGoogle Scholar
  34. 34.
    Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797. doi: 10.1093/nar/gkh340 CrossRefGoogle Scholar
  35. 35.
    Galtier N, Gouy M, Gautier C (1996) SEAVIEW and PHYLO_WIN: two graphic tools for sequence alignment and molecular phylogeny. Bioinformatics 12:543–548. doi: 10.1093/bioinformatics/12.6.543 CrossRefGoogle Scholar
  36. 36.
    Rost B, Fariselli P, Casadio R (1996) Topology prediction for helical transmembrane proteins at 86% accuracy. Protein Sci 5:1704–1718. doi: 10.1002/pro.5560050824 CrossRefGoogle Scholar
  37. 37.
    Juretic D, Zoranic L, Zucic D (2002) Basic charge clusters and predictions of membrane protein topology. J Chem Inf Comp Sci 42:620–632. doi: 10.1021/ci010263s Google Scholar
  38. 38.
    Tusnády GE, Simon I (2001) The HMMTOP transmembrane topology prediction server. Bioinformatics 17:849–850. doi: 10.1093/bioinformatics/17.9.849 CrossRefGoogle Scholar
  39. 39.
    Krogh A, Larsson B, von Heijne G, Sonnhammer ELL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580. doi: 10.1006/jmbi.2000.4315 CrossRefGoogle Scholar
  40. 40.
    Kahsay RY, Gao G, Liao L (2005) An improved hidden Markov model for transmembrane protein detection and topology prediction and its applications to complete genomes. Bioinformatics 21:1853–1858. doi: 10.1093/bioinformatics/bti303 CrossRefGoogle Scholar
  41. 41.
    Park Y, Helms V (2008) Prediction of the translocon-mediated membrane insertion free energies of protein sequences. Bioinformatics 24:1271–1277. doi: 10.1093/bioinformatics/btn114 CrossRefGoogle Scholar
  42. 42.
    Canutescu AA, Shelenkov AA, Dunbrack RL (2003) A graph-theory algorithm for rapid protein side-chain prediction. Protein Sci 12:2001–2014. doi: 10.1110/ps.03154503 CrossRefGoogle Scholar
  43. 43.
    Hess B, Kutzner C, van der Spoel D, Lindahl E (2008) GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput 4:435–447. doi: 10.1021/ct700301q CrossRefGoogle Scholar
  44. 44.
    Griepernau B, Leis S, Schneider MF, Sikor M, Steppich D, Bockmann RA (2007) 1-Alkanols and membranes: a story of attraction. Biochim Biophys Acta 1768:2899–2913. doi: 10.1016/j.bbamem.2007.08.002 CrossRefGoogle Scholar
  45. 45.
    Faraldo-Gómez JD, Smith GR, Sansom MS (2002) Setting up and optimization of membrane protein simulations. Eur Biophys J 31:217–227. doi: 10.1007/s00249-002-0207-5 CrossRefGoogle Scholar
  46. 46.
    Sanner MF, Olson AJ, Spehner JC (1996) Reduced surface: an efficient. Biopolymers 38:305–320. doi: 10.1002/(SICI)1097-0282(199603)38:3<305::AID-BIP4>3.0.CO;2-Y CrossRefGoogle Scholar
  47. 47.
    Berendsen HJC, van der Spoel D, van Drunen R (1995) Gromacs-a message-passing parallel molecular-dynamics implementation. Comput Phys Commun 91:43–56. doi: 10.1016/0010-4655(95)00042-E CrossRefGoogle Scholar
  48. 48.
    Siu SWI, Vácha R, Jungwirth P, Böckmann RA (2008) Biomolecular simulations of membranes: physical properties from different force fields. J Chem Phys 128:125103. doi: 10.1063/1.2897760 CrossRefGoogle Scholar
  49. 49.
    Oostenbrink C, Villa A, Mark AE, van Gunsteren WF (2004) A biomolecular force field based on the free enthalpy of hydration and solvation: the GROMOS force-field parameter sets 53A5 and 53A6. J Comput Chem 25:1656–1676. doi: 10.1002/jcc.20090 CrossRefGoogle Scholar
  50. 50.
    Berger O, Edholm O, Jahnig F (1997) Molecular dynamics simulations of a fluid bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and constant temperature. Biophys J 72:2002–2013. doi: 10.1016/S0006-3495(97)78845-3 CrossRefGoogle Scholar
  51. 51.
    Chiu SW, Clark M, Balaji V, Subramaniam S, Scott HL, Jakobsson E (1995) Incorporation of surface tension into molecular dynamics simulation of an interface: a fluid phase lipid bilayer membrane. Biophys J 69:1230–1245. doi: 10.1016/S0006-3495(95)80005-6 CrossRefGoogle Scholar
  52. 52.
    Berendsen HJC, Postma JPM, van Gunsteren WF, DiNola A, Haak JR (1984) Molecular dynamics with coupling to an external bath. J Chem Phys 81:3684. doi: 10.1063/1.448118 CrossRefGoogle Scholar
  53. 53.
    Hess B, Bekker H, Berendsen HJC, Fraaije JGEM (1997) LINCS: a linear constraint solver for molecular simulations. J Comput Chem 18:1463–1472. doi: 10.1002/(SICI)1096-987X(199709)18:12<1463::AID-JCC4>3.0.CO;2-H CrossRefGoogle Scholar
  54. 54.
    Park Y, Helms V (2008) MINS2: revisiting the molecular code for transmembrane-helix recognition by the Sec61 translocon. Bioinformatics 24:1819–1820. doi: 10.1093/bioinformatics/btn255 CrossRefGoogle Scholar
  55. 55.
    De Beeck AO et al. (2000) The transmembrane domains of hepatitis C virus envelope glycoproteins E1 and E2 play a major role in heterodimerization. J Biol Chem 275:31428–31437. doi: 10.1074/jbc.M003003200 CrossRefGoogle Scholar
  56. 56.
    Johansson ACV, Lindahl E (2006) Amino-acid solvation structure in transmembrane helices from molecular dynamics simulations. Biophys J 91:4450–4463. doi: 10.1529/biophysj.106.092767 CrossRefGoogle Scholar
  57. 57.
    Bonifacino JS, Cosson P, Shah N, Klausner RD (1991) Role of potentially charged transmembrane residues in targeting proteins for retention and degradation within the endoplasmic reticulum. EMBO J 1991:2783–2793Google Scholar
  58. 58.
    Freites JA, Tobias DJ, von Heijne G, White SH (2005) Interface connections of a transmembrane voltage sensor. Proc Natl Acad Sci USA 102:15059–15064. doi: 10.1073/pnas.0507618102 CrossRefGoogle Scholar
  59. 59.
    Joh NHJ et al. (2008) Modest stabilization by most hydrogen-bonded side-chain interactions in membrane proteins. Nature 453:1266–1270. doi: 10.1038/nature06977 CrossRefGoogle Scholar
  60. 60.
    Meindl-Beinker NM, Lundin C, Nilsson IM, White SH, von Heijne G (2006) Asn-and Asp-mediated interactions between transmembrane helices during translocon-mediated membrane protein assembly. EMBO Rep 7:1111. doi: 10.1038/sj.embor.7400818 CrossRefGoogle Scholar
  61. 61.
    Im W, Feig M, Brooks CL (2003) An implicit membrane generalized Born theory for the study of structure, stability, and interactions of membrane proteins. Biophys J 85:2900–2918. doi: 10.1016/S0006-3495(03)74712-2 CrossRefGoogle Scholar
  62. 62.
    Tanizaki S, Feig M (2005) A generalized Born formalism for heterogeneous dielectric environments: application to the implicit modeling of biological membranes. J Chem Phys 122:124706. doi: 10.1063/1.1865992 CrossRefGoogle Scholar
  63. 63.
    Bu L, Brooks RCL (2008) De novo prediction of the structures of M. tuberculosis membrane proteins. J Am Chem Soc 130:5384–5385. doi: 10.1021/ja710213p CrossRefGoogle Scholar
  64. 64.
    Bu L, Im W, Brooks C (2007) Membrane assembly of simple helix homo-oligomers studied via molecular dynamics simulations. Biophys J 92:854–863. doi: 10.1529/biophysj.106.095216 CrossRefGoogle Scholar
  65. 65.
    Lazaridis T (2003) Effective energy function for proteins in lipid membranes. Proteins 52:176–192. doi: 10.1002/prot.10410 CrossRefGoogle Scholar
  66. 66.
    Mottamal M, Zhang J, Lazaridis T (2006) Energetics of the native and non-native states of the glycophorin transmembrane helix dimer. Proteins 62. doi:10.1002/prot.20844Google Scholar
  67. 67.
    Ulmschneider M, Ulmschneider J, Sansom M, Di Nola A (2007) A generalized born implicit-membrane representation compared to experimental insertion free energies. Biophys J 92:2338–2349. doi: 10.1529/biophysj.106.081810 CrossRefGoogle Scholar
  68. 68.
    Nymeyer H, Woolf TB, Garcia AE (2005) Folding is not required for bilayer insertion: replica exchange simulations of an a-helical peptide with an explicit lipid bilayer. Proteins 59:783–790. doi: 10.1002/prot.20460 CrossRefGoogle Scholar
  69. 69.
    Benedix A, Becker CM, de Groot BL, Caflisch A, Bockmann RA (2009) Predicting free energy changes using structural ensembles. Nat Meth 6:3–4. doi: 10.1038/nmeth0109-3 CrossRefGoogle Scholar
  70. 70.
    Potapov V, Cohen M, Schreiber G (2009) Assessing computational methods for predicting protein stability upon mutation: good on average but not in the details. Protein Eng Des Sel 22:553–560. doi: 10.1093/protein/gzp030 CrossRefGoogle Scholar
  71. 71.
    Langosch D, Brosig B, Kolmar H, Fritz HJ (1996) Dimerisation of the glycophorin A transmembrane segment in membranes probed with the ToxR transcription activator. J Mol Biol 263:525–530. doi: 10.1006/jmbi.1996.0595 CrossRefGoogle Scholar
  72. 72.
    Senes A, Gerstein M, Engelman DM (2000) Statistical analysis of amino acid patterns in transmembrane helices: the GxxxG motif occurs frequently and in association with ß-branched residues at neighboring positions. J Mol Biol 296:921–936. doi: 10.1006/jmbi.1999.3488 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Siti Azma Jusoh
    • 1
    • 2
  • Christoph Welsch
    • 3
  • Shirley W. I. Siu
    • 4
  • Rainer A. Böckmann
    • 4
  • Volkhard Helms
    • 1
    Email author
  1. 1.Center for BioinformaticsSaarland UniversitySaarbrueckenGermany
  2. 2.Faculty of PharmacyUniversiti Technologi MARAShah AlamMalaysia
  3. 3.Department of Internal Medicine IJohann Wolfgang Goethe-University HospitalFrankfurt am MainGermany
  4. 4.Computational BiologyUniversität Erlangen-Nürnberg, BTE GebäudeErlangenGermany

Personalised recommendations