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.

A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex

This article has been updated

Abstract

Most transmembrane proteins are selected as transport-vesicle cargo through the recognition of short, linear amino-acid motifs in their cytoplasmic portions by vesicle coat proteins. For clathrin-coated vesicles, the motifs are recognized by clathrin adaptors. The AP2 adaptor complex (subunits α, β2, μ2 and σ2) recognizes both major endocytic motifs: YxxΦ motifs1 (where Φ can be F, I, L, M or V) and [ED]xxxL[LI] acidic dileucine motifs. Here we describe the binding of AP2 to the endocytic dileucine motif from CD4 (ref. 2). The major recognition events are the two leucine residues binding in hydrophobic pockets on σ2. The hydrophilic residue four residues upstream from the first leucine sits on a positively charged patch made from residues on the σ2 and α subunits. Mutations in key residues inhibit the binding of AP2 to ‘acidic dileucine’ motifs displayed in liposomes containing phosphatidylinositol-4,5-bisphosphate, but do not affect binding to YxxΦ motifs through μ2. In the ‘inactive’ AP2 core structure3 both motif-binding sites are blocked by different parts of the β2 subunit. To allow a dileucine motif to bind, the β2 amino terminus is displaced and becomes disordered; however, in this structure the YxxΦ-binding site on μ2 remains blocked.

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

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Structure of the AP2 adaptor core in complex with the dileucine peptide from CD4.
Figure 2: Details of binding of the CD4 dileucine signal by the σ2 and α subunits of AP2.
Figure 3: Confirmation of location and conservation among different σ subunits of the dileucine-motif-binding site.
Figure 4: A conformational change in AP2 is required for dileucine peptide binding.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited with the Protein Data Bank under accession numbers 2jkr for the Q peptide complex and 2jkt for the E peptide complex, respectively.

Change history

  • 18 December 2008

    The AOP version of this paper carried an erroneous affiliation for D.J.O. This was corrected on 18 December 2008.

References

  1. Owen, D. J. & Evans, P. R. A structural explanation for the recognition of tyrosine-based endocytotic signals. Science 282, 1327–1332 (1998)

    ADS  CAS  Article  Google Scholar 

  2. Pitcher, C., Honing, S., Fingerhut, A., Bowers, K. & Marsh, M. Cluster of differentiation antigen 4 (CD4) endocytosis and adaptor complex binding require activation of the CD4 endocytosis signal by serine phosphorylation. Mol. Biol. Cell 10, 677–691 (1999)

    CAS  Article  Google Scholar 

  3. Collins, B. M., McCoy, A. J., Kent, H. M., Evans, P. R. & Owen, D. J. Molecular architecture and functional model of the endocytic AP2 complex. Cell 109, 523–535 (2002)

    CAS  Article  Google Scholar 

  4. Hurley, J. H., Lee, S. & Prag, G. Ubiquitin-binding domains. Biochem. J. 399, 361–372 (2006)

    CAS  Article  Google Scholar 

  5. Pryor, P. R. et al. Molecular basis for the sorting of the SNARE VAMP7 into endocytic clathrin-coated vesicles by the ArfGAP Hrb. Cell 134, 817–827 (2008)

    CAS  Article  Google Scholar 

  6. Miller, S. E., Collins, B. M., McCoy, A. J., Robinson, M. S. & Owen, D. J. A SNARE-adaptor interaction is a new mode of cargo recognition in clathrin-coated vesicles. Nature 450, 570–574 (2007)

    ADS  CAS  Article  Google Scholar 

  7. Bonifacino, J. S. & Traub, L. M. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72, 395–447 (2003)

    CAS  Article  Google Scholar 

  8. Shiba, T. et al. Structural basis for recognition of acidic-cluster dileucine sequence by GGA1. Nature 415, 937–941 (2002)

    ADS  CAS  Article  Google Scholar 

  9. Misra, S., Puertollano, R., Kato, Y., Bonifacino, J. S. & Hurley, J. H. Structural basis for acidic-cluster-dileucine sorting-signal recognition by VHS domains. Nature 415, 933–937 (2002)

    ADS  CAS  Article  Google Scholar 

  10. Chaudhuri, R., Lindwasser, O. W., Smith, W. J., Hurley, J. H. & Bonifacino, J. S. Downregulation of CD4 by human immunodeficiency virus type 1 Nef is dependent on clathrin and involves direct interaction of Nef with the AP2 clathrin adaptor. J. Virol. 81, 3877–3890 (2007)

    CAS  Article  Google Scholar 

  11. Doray, B., Lee, I., Knisely, J., Bu, G. & Kornfeld, S. The γ/σ1 and α/σ2 hemicomplexes of clathrin adaptors AP-1 and AP-2 harbor the dileucine recognition site. Mol. Biol. Cell 18, 1887–1896 (2007)

    CAS  Article  Google Scholar 

  12. Honing, 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 

  13. Letourneur, F. & Klausner, R. D. A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains. Cell 69, 1143–1157 (1992)

    CAS  Article  Google Scholar 

  14. Doray, B., Knisely, J. M., Wartman, L., Bu, G. & Kornfeld, S. Identification of acidic dileucine signals in LRP9 that interact with both GGAs and AP-1/AP-2. Traffic (in the press)

  15. Huang, F., Jiang, X. & Sorkin, A. Tyrosine phosphorylation of the β2 subunit of clathrin adaptor complex AP-2 reveals the role of a di-leucine motif in the epidermal growth factor receptor trafficking. J. Biol. Chem. 278, 43411–43417 (2003)

    CAS  Article  Google Scholar 

  16. Lee, I., Doray, B., Govero, J. & Kornfeld, S. Binding of cargo sorting signals to AP-1 enhances its association with ADP ribosylation factor 1-GTP. J. Cell Biol. 180, 467–472 (2008)

    CAS  Article  Google Scholar 

  17. Mancias, J. D. & Goldberg, J. The transport signal on Sec22 for packaging into COPII-coated vesicles is a conformational epitope. Mol. Cell 26, 403–414 (2007)

    CAS  Article  Google Scholar 

  18. Schwartz, T. & Blobel, G. Structural basis for the function of the β subunit of the eukaryotic signal recognition particle receptor. Cell 112, 793–803 (2003)

    CAS  Article  Google Scholar 

  19. Bethune, J. et al. Coatomer, the coat protein of COPI transport vesicles, discriminates endoplasmic reticulum residents from p24 proteins. Mol. Cell. Biol. 26, 8011–8021 (2006)

    CAS  Article  Google Scholar 

  20. McCoy, A. J., Grosse-Kunstleve, R. W., Storoni, L. C., Adams, P. D. & Read, R. J. PHASER crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    CAS  Article  Google Scholar 

  21. Leslie, A. G. The integration of macromolecular diffraction data. Acta Crystallogr. D 62, 48–57 (2006)

    Article  Google Scholar 

  22. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006)

    Article  Google Scholar 

  23. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

  24. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  25. Painter, J. & Merritt, E. A. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D 62, 439–450 (2006)

    Article  Google Scholar 

  26. Ricotta, D., Conner, S. D., Schmid, S. L., von Figura, K. & Honing, S. Phosphorylation of the AP2 μ subunit by AAK1 mediates high affinity binding to membrane protein sorting signals. J. Cell Biol. 156, 791–795 (2002)

    CAS  Article  Google Scholar 

  27. Jonsson, U. et al. Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. Biotechniques 11, 620–627 (1991)

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank the protein crystallography beamline staff at Diamond, especially E. Duke, K. McAuley and R. Flaig, for their support and assistance. D.J.O., B.T.K. and S.E.M. are funded by a Wellcome Trust Senior Research Fellowship to D.J.O. S.H. and K.S. are supported by grants from the Deutsche Forschungsgemeinschaft (SFB635 and SFB670).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to David J. Owen.

Supplementary information

Supplementary Information

This file contains Supplementary Figures S1-S9 with Legends and Supplementary Tables S1-S3. (PDF 2219 kb)

Supplementary Movie 1

Supplementary Movie 1 shows animation of the conformational change between the IP6-liganded, inactive conformation (2vgl) and the acidic dileucine motif bound conformations of the AP2 core. The two structures were superimposed on the σ2 subunit. Residues Y6 and F7 are highlighted in the closed structure (green balls) and the dileucine peptide is shown in gold. The two structures are alternated first in a "front" view, then from a "back" view, rotated by 180° (MOV 2055 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kelly, B., McCoy, A., Späte, K. et al. A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex. Nature 456, 976–979 (2008). https://doi.org/10.1038/nature07422

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature07422

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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