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

Nature Chemical Biology volume 6, pages 4653 (2010) | Download Citation

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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.

  • Compound C14H28O2

    Myristate

  • Compound C45H68NO8P

    2-(3-(Diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine

  • Compound C44H84NO8P

    1,2-Dioleoyl-sn-glycero-3-phosphocholine

  • Compound C42H78NaO10P

    1,2-Dioleoyl-sn-glycero-3-phospho(1-rac-glycerol)

  • Compound C41H78NO8P

    1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine

  • Compound C42H77NNaO10P

    1,2-Dioleoyl-sn-glycero-3-[phospho-L-serine]

  • Compound C45H94N3O19P3

    Phosphatidylinositol-4,5-bisphosphate

  • Compound C27H46O

    Cholesterol

  • Compound C41H83N2O6P

    Sphingomyelin

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References

  1. 1.

    & Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature 438, 590–596 (2005).

  2. 2.

    & Plasma membrane phosphoinositide organization by protein electrostatics. Nature 438, 605–611 (2005).

  3. 3.

    & Role of cholesterol and lipid organization in disease. Nature 438, 612–621 (2005).

  4. 4.

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

  5. 5.

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

  6. 6.

    , , , & 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).

  7. 7.

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

  8. 8.

    , & Structure-function relationships in HIV-1 Nef. EMBO Rep. 2, 580–585 (2001).

  9. 9.

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

  10. 10.

    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).

  11. 11.

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

  12. 12.

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

  13. 13.

    , & 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).

  14. 14.

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

  15. 15.

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

  16. 16.

    & Roles of bilayer material properties in function and distribution of membrane proteins. Annu. Rev. Biophys. Biomol. Struct. 35, 177–198 (2006).

  17. 17.

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

  18. 18.

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

  19. 19.

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

  20. 20.

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

  21. 21.

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

  22. 22.

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

  23. 23.

    , , , & ArfGAP1 responds to membrane curvature through the folding of a lipid packing sensor motif. EMBO J. 24, 2244–2253 (2005).

  24. 24.

    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).

  25. 25.

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

  26. 26.

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

  27. 27.

    , , & 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).

  28. 28.

    & How proteins adapt to a membrane-water interface. Trends Biochem. Sci. 25, 429–434 (2000).

  29. 29.

    & Experimentally determined hydrophobicity scale for proteins at membrane interfaces. Nat. Struct. Biol. 3, 842–848 (1996).

  30. 30.

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

  31. 31.

    , , , & Structure of the anchor-domain of myristoylated and non-myristoylated HIV-1 Nef protein. J. Mol. Biol. 289, 123–138 (1999).

  32. 32.

    , , , & Solution structure of a polypeptide from the N terminus of the HIV protein Nef. Biochemistry 36, 5970–5980 (1997).

  33. 33.

    , , & 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).

  34. 34.

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

  35. 35.

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

  36. 36.

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

  37. 37.

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

  38. 38.

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

  39. 39.

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

  40. 40.

    & 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).

  41. 41.

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

  42. 42.

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

  43. 43.

    , , , & Synaptotagmin activates membrane fusion through a Ca2+-dependent trans interaction with phospholipids. Nat. Struct. Mol. Biol. 14, 904–911 (2007).

  44. 44.

    , & How Synaptotagmin promotes membrane fusion. Science 316, 1205–1208 (2007).

  45. 45.

    , , , & Cryo-electron tomography of clathrin-coated vesicles: structural implications for coat assembly. J. Mol. Biol. 365, 892–899 (2007).

  46. 46.

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

  47. 47.

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

  48. 48.

    , & Extrusion technique to generate liposomes of defined size. Methods Enzymol. 367, 3–14 (2003).

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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

    • Christian F W Becker

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

Affiliations

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

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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.

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DOI

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

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