Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

HIV-1 Nef membrane association depends on charge, curvature, composition and sequence

Nature Chemical Biology volume 6, pages 46–53 (2010)Cite this article

Abstract

Nef-mediated internalization of T-cell receptor molecules from the surface of an infected cell is required for the pathogenicity of HIV and disease progression to AIDS. This function depends on the N-terminal myristoylation of Nef, a lipid modification that targets the protein to membranes. We have analyzed how specific membrane properties and sequence motifs within Nef determine this interaction. Using time-resolved techniques we find that the association with membranes is a biphasic process with a fast rate for an electrostatic-driven protein-liposome interaction and a slow rate for the formation of an amphipathic helix. The rate of myristate insertion into liposomes depends on membrane curvature, while changes in the lipid composition with respect to phosphoinositides, cholesterol or sphingomyelin did not significantly alter the interaction. Moreover, Nef binding to membranes requires negatively charged liposomes, and mutations of basic and hydrophobic residues strongly diminished the association and changed the binding kinetics differently.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Nef binding to lipid membranes is a biphasic process.
Figure 2: An acidic membrane surface is required for Nef binding.
Figure 3: Membrane association of Nef correlates with liposome curvature.
Figure 4: N-terminal residues in Nef sustain the membrane association.
Figure 5: Kinetics of Nef mutants revealed different contributions of basic and hydrophobic residues for the two phases of membrane binding.
Figure 6: Kinetic model for Nef membrane binding.

Similar content being viewed by others

References

  1. McMahon, H.T. & Gallop, J.L. Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature 438, 590–596 (2005).

    Article  CAS  Google Scholar 

  2. McLaughlin, S. & Murray, D. Plasma membrane phosphoinositide organization by protein electrostatics. Nature 438, 605–611 (2005).

    Article  CAS  Google Scholar 

  3. Maxfield, F.R. & Tabas, I. Role of cholesterol and lipid organization in disease. Nature 438, 612–621 (2005).

    Article  CAS  Google Scholar 

  4. Stevenson, M. HIV-1 pathogenesis. Nat. Med. 9, 853–860 (2003).

    Article  CAS  Google Scholar 

  5. Deacon, N.J. et al. Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients. Science 270, 988–991 (1995).

    Article  CAS  Google Scholar 

  6. Kirchhoff, F., Greenough, T.C., Brettler, D.B., Sullivan, J.L. & Desrosiers, R.C. Brief report: absence of intact nef sequences in a long-term survivor with nonprogressive HIV-1 infection. N. Engl. J. Med. 332, 228–232 (1995).

    Article  CAS  Google Scholar 

  7. Greene, W.C. & Peterlin, B.M. Charting HIV's remarkable voyage through the cell: basic science as a passport to future therapy. Nat. Med. 8, 673–680 (2002).

    Article  CAS  Google Scholar 

  8. Geyer, M., Fackler, O.T. & Peterlin, B.M. Structure-function relationships in HIV-1 Nef. EMBO Rep. 2, 580–585 (2001).

    Article  CAS  Google Scholar 

  9. Malim, M.H. & Emerman, M. HIV-1 accessory proteins–ensuring viral survival in a hostile environment. Cell Host Microbe 3, 388–398 (2008).

    Article  CAS  Google Scholar 

  10. Schindler, M. et al. Nef-mediated suppression of T cell activation was lost in a lentiviral lineage that gave rise to HIV-1. Cell 125, 1055–1067 (2006).

    Article  CAS  Google Scholar 

  11. Resh, M.D. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim. Biophys. Acta 1451, 1–16 (1999).

    Article  CAS  Google Scholar 

  12. Giese, S.I. et al. Specific and distinct determinants mediate membrane binding and lipid raft incorporation of HIV-1(SF2) Nef. Virology 355, 175–191 (2006).

    Article  CAS  Google Scholar 

  13. Bentham, M., Mazaleyrat, S. & Harris, M. Role of myristoylation and N-terminal basic residues in membrane association of the human immunodeficiency virus type 1 Nef protein. J. Gen. Virol. 87, 563–571 (2006).

    Article  CAS  Google Scholar 

  14. Fackler, O.T. et al. Functional characterization of HIV-1 Nef mutants in the context of viral infection. Virology 351, 322–339 (2006).

    Article  CAS  Google Scholar 

  15. Breuer, S. et al. Biochemical indication for myristoylation-dependent conformational changes in HIV-1 Nef. Biochemistry 45, 2339–2349 (2006).

    Article  CAS  Google Scholar 

  16. McIntosh, T.J. & Simon, S.A. Roles of bilayer material properties in function and distribution of membrane proteins. Annu. Rev. Biophys. Biomol. Struct. 35, 177–198 (2006).

    Article  CAS  Google Scholar 

  17. Yeung, T. et al. Membrane phosphatidylserine regulates surface charge and protein localization. Science 319, 210–213 (2008).

    Article  CAS  Google Scholar 

  18. Frost, A. et al. Structural basis of membrane invagination by F-BAR domains. Cell 132, 807–817 (2008).

    Article  CAS  Google Scholar 

  19. Gallop, J.L. et al. Mechanism of endophilin N-BAR domain-mediated membrane curvature. EMBO J. 25, 2898–2910 (2006).

    Article  CAS  Google Scholar 

  20. Peter, B.J. et al. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303, 495–499 (2004).

    Article  CAS  Google Scholar 

  21. Ford, M.G. et al. Curvature of clathrin-coated pits driven by epsin. Nature 419, 361–366 (2002).

    Article  CAS  Google Scholar 

  22. Drin, G. et al. A general amphipathic alpha-helical motif for sensing membrane curvature. Nat. Struct. Mol. Biol. 14, 138–146 (2007).

    Article  CAS  Google Scholar 

  23. Bigay, J., Casella, J.-F., Drin, G., Mesmin, B. & Antonny, B. ArfGAP1 responds to membrane curvature through the folding of a lipid packing sensor motif. EMBO J. 24, 2244–2253 (2005).

    Article  CAS  Google Scholar 

  24. Heo, W.D. et al. PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane. Science 314, 1458–1461 (2006).

    Article  CAS  Google Scholar 

  25. Rocks, O. et al. An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science 307, 1746–1752 (2005).

    Article  CAS  Google Scholar 

  26. Brügger, B. et al. Human immunodeficiency virus type 1 Nef protein modulates the lipid composition of virions and host cell membrane microdomains. Retrovirology 4, 70 (2007).

    Article  Google Scholar 

  27. Welker, R., Harris, M., Cardel, B. & Kräusslich, H.G. Virion incorporation of human immunodeficiency virus type 1 Nef is mediated by a bipartite membrane-targeting signal: analysis of its role in enhancement of viral infectivity. J. Virol. 72, 8833–8840 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Killian, J.A. & von Heijne, G. How proteins adapt to a membrane-water interface. Trends Biochem. Sci. 25, 429–434 (2000).

    Article  CAS  Google Scholar 

  29. Wimley, W.C. & White, S.H. Experimentally determined hydrophobicity scale for proteins at membrane interfaces. Nat. Struct. Biol. 3, 842–848 (1996).

    Article  CAS  Google Scholar 

  30. Geyer, M. & Peterlin, B.M. Domain assembly, surface accessibility and sequence conservation in full length HIV-1 Nef. FEBS Lett. 496, 91–95 (2001).

    Article  CAS  Google Scholar 

  31. Geyer, M., Munte, C.E., Schorr, J., Kellner, R. & Kalbitzer, H.R. Structure of the anchor-domain of myristoylated and non-myristoylated HIV-1 Nef protein. J. Mol. Biol. 289, 123–138 (1999).

    Article  CAS  Google Scholar 

  32. Barnham, K.J., Monks, S.A., Hinds, M.G., Azad, A.A. & Norton, R.S. Solution structure of a polypeptide from the N terminus of the HIV protein Nef. Biochemistry 36, 5970–5980 (1997).

    Article  CAS  Google Scholar 

  33. Antonny, B., Beraud-Dufour, S., Chardin, P. & Chabre, M. N-terminal hydrophobic residues of the G-protein ADP-ribosylation factor-1 insert into membrane phospholipids upon GDP to GTP exchange. Biochemistry 36, 4675–4684 (1997).

    Article  CAS  Google Scholar 

  34. Brügger, B. et al. The HIV lipidome: a raft with an unusual composition. Proc. Natl. Acad. Sci. USA 103, 2641–2646 (2006).

    Article  Google Scholar 

  35. Resh, M.D. Trafficking and signaling by fatty-acylated and prenylated proteins. Nat. Chem. Biol. 2, 584–590 (2006).

    Article  CAS  Google Scholar 

  36. Ames, J.B. et al. Molecular mechanics of calcium-myristoyl switches. Nature 389, 198–202 (1997).

    Article  CAS  Google Scholar 

  37. McLaughlin, S. & Aderem, A. The myristoyl-electrostatic switch: a modulator of reversible protein-membrane interactions. Trends Biochem. Sci. 20, 272–276 (1995).

    Article  CAS  Google Scholar 

  38. Tang, C. et al. Entropic switch regulates myristate exposure in the HIV-1 matrix protein. Proc. Natl. Acad. Sci. USA 101, 517–522 (2004).

    Article  CAS  Google Scholar 

  39. Höning, S. et al. Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2. Mol. Cell 18, 519–531 (2005).

    Article  Google Scholar 

  40. Dries, D.R. & Newton, A.C. Kinetic analysis of the interaction of the C1 domain of protein kinase C with lipid membranes by stopped-flow spectroscopy. J. Biol. Chem. 283, 7885–7893 (2008).

    Article  CAS  Google Scholar 

  41. Nalefski, E.A. & Newton, A.C. Membrane binding kinetics of protein kinase C betaII mediated by the C2 domain. Biochemistry 40, 13216–13229 (2001).

    Article  CAS  Google Scholar 

  42. Arbuzova, A. et al. Kinetics of interaction of the myristoylated alanine-rich C kinase substrate, membranes, and calmodulin. J. Biol. Chem. 272, 27167–27177 (1997).

    Article  CAS  Google Scholar 

  43. Stein, A., Radhakrishnan, A., Riedel, D., Fasshauer, D. & Jahn, R. Synaptotagmin activates membrane fusion through a Ca2+-dependent trans interaction with phospholipids. Nat. Struct. Mol. Biol. 14, 904–911 (2007).

    Article  CAS  Google Scholar 

  44. Martens, S., Kozlov, M.M. & McMahon, H.T. How Synaptotagmin promotes membrane fusion. Science 316, 1205–1208 (2007).

    Article  CAS  Google Scholar 

  45. Cheng, Y., Boll, W., Kirchhausen, T., Harrison, S.C. & Walz, T. Cryo-electron tomography of clathrin-coated vesicles: structural implications for coat assembly. J. Mol. Biol. 365, 892–899 (2007).

    Article  CAS  Google Scholar 

  46. Takamori, S. et al. Molecular anatomy of a trafficking organelle. Cell 127, 831–846 (2006).

    Article  CAS  Google Scholar 

  47. Zimmerberg, J. & Kozlov, M.M. How proteins produce cellular membrane curvature. Nat. Rev. Mol. Cell Biol. 7, 9–19 (2006).

    Article  CAS  Google Scholar 

  48. Mui, B., Chow, L. & Hope, M.J. Extrusion technique to generate liposomes of defined size. Methods Enzymol. 367, 3–14 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank S. Gentz (Max Planck Institute for Molecular Physiology) for peptide synthesis, G. Holtermann for expert technical support and O. Fackler for stimulating discussions. F. Thorwirth, P. Verveer and P. Bastiaens are kindly acknowledged for help and advice with fluorescence imaging.

Author information

Author notes

  1. Christian F W Becker

    Present address: Present address: Department of Chemistry, Technische Universität München, Lichtenbergstrasse 4, Garching, Germany.,

Authors and Affiliations

  1. Abteilung Physikalische Biochemie, Max-Planck-Institut für molekulare Physiologie, Dortmund, Germany

    Holger Gerlach, Vanessa Laumann, Christian F W Becker, Roger S Goody & Matthias Geyer

  2. Max F. Perutz Laboratories, University of Vienna, Vienna, Austria

    Sascha Martens

Authors
  1. Holger Gerlach
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vanessa Laumann
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sascha Martens
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christian F W Becker
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Roger S Goody
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Matthias Geyer
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.G. performed most of the experiments with support of V.L. and C.F.W.B.; S.M. contributed to tubulation experiments. R.S.G. supervised the kinetic data analyses. M.G. designed the study and wrote the manuscript together with R.S.G., with the support of S.M. and C.F.W.B. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Matthias Geyer.

Supplementary information

Supplementary Text and Figures

Supplementary Results and Supplementary Methods (PDF 4778 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gerlach, H., Laumann, V., Martens, S. et al. HIV-1 Nef membrane association depends on charge, curvature, composition and sequence. Nat Chem Biol 6, 46–53 (2010). https://doi.org/10.1038/nchembio.268

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.268

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing