Letters to Nature

Nature , | doi:10.1038/nature07422; Received 8 July 2008; Accepted 4 September 2008; Published online 2 November 2008

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

Bernard T. Kelly1,4, Airlie J. McCoy1,4, Kira Späte2,5, Sharon E. Miller1, Philip R. Evans3, Stefan Höning2 & David J. Owen1,5

  1. Cambridge Institute for Medical Research and Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 0XY, UK
  2. Institute of Biochemistry I and Center for Molecular Medicine Cologne, University of Cologne, Joseph-Stelzmann-Strasse 52, 50931 Cologne, Germany
  3. Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK
  4. These authors contributed equally to this work.
  5. Present address: Max Planck Institute for Experimental Medicine, Hermann-Rein-Strasse 3, 37075 Göttingen, Germany.

Correspondence to: David J. Owen1,5 Correspondence and requests for materials should be addressed to D.J.O. (Email: djo30@cam.ac.uk).

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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 alpha, beta2, mu2 and sigma2) recognizes both major endocytic motifs: YxxPhi motifs1 (where Phi 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 sigma2. The hydrophilic residue four residues upstream from the first leucine sits on a positively charged patch made from residues on the sigma2 and alpha 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 YxxPhi motifs through mu2. In the 'inactive' AP2 core structure3 both motif-binding sites are blocked by different parts of the beta2 subunit. To allow a dileucine motif to bind, the beta2 amino terminus is displaced and becomes disordered; however, in this structure the YxxPhi-binding site on mu2 remains blocked.

In clathrin-coated vesicles (CCVs), cargo is selected mainly through the recognition of one of three types of signal by clathrin adaptors: the short, linear, transplantable peptide motifs that are widely found on general cargo; covalently attached ubiquitin molecules (reviewed in ref. 4); and the folded determinants on SNAP receptor (SNARE) proteins5, 6. The two major classes of transplantable motif used in CCVs are the YxxPhi and acidic dileucine motifs. The latter can be divided into two further classes: the [ED]xxxL[LI] motifs, which, along with YxxPhi motifs, are recognized by the heterotetrameric AP clathrin adaptor complexes on numerous different pathways (reviewed in ref. 7), and the DxxLL motifs, recognized by GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-interacting proteins) for transport from the trans-Golgi network to endosomes8, 9. AP2 has a pivotal function in regulating the formation of endocytic CCVs destined for early endosomes. Although the mechanism of YxxPhi motif binding by APs has been characterized at the molecular level, there is no mechanistic insight into [ED]xxxL[LI] binding, although several recent studies have indicated that the binding site resides on the alpha–sigma2 heterodimer10, 11. Eight different 'acidic dileucine' peptides were tried in co-crystallization experiments with the 200-kDa AP2 core, and several produced poorly diffracting crystals. However, co-crystallization with the peptide RM(phosphoS)QIKRLLSE (Q peptide) from the T-cell cell-surface antigen protein CD4, and a variant of this peptide (RM(phosphoS)EIKRLLSE) (E peptide), resulted in crystals that diffracted to resolutions of 3.0 and 3.4 Å respectively. The structure of the Q peptide complex was solved by molecular replacement by using models derived from the closed structure3 (Fig. 1). Rebuilding of the alpha and beta2 helical solenoids was required because of alterations in their overall curvature (see below), but even in the initial electron density maps the 'dileucine peptide' could be seen clearly. The identity and orientation of the peptide was confirmed as described in Methods.

Figure 1: Structure of the AP2 adaptor core in complex with the dileucine peptide from CD4.
Figure 1 : Structure of the AP2 adaptor core in complex with the dileucine peptide from CD4. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

Orthogonal views of the dileucine motif liganded AP2 complex in ribbon (a, b) and in molecular surface (c, d) representations. In a and c the membrane is parallel to the upper face of the complex, and in b and d the complex is viewed through the membrane, with the membrane interacting surface facing upwards. The alpha subunit is coloured dark blue, beta2 green, sigma2 pale blue, N-mu2 purple and C-mu2 mauve. Atoms in the the dileucine motif peptide are shown as spheres, with carbons coloured gold. The two sulphate groups bound to the alpha subunit in the PtdIns(4,5)P2 site are shown.

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The structure shows that the peptide binds in an extended conformation near the alpha-subunit binding site for phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2), which is replaced here by two sulphate ions (Fig. 1 and Supplementary Fig. 3). The LL moiety at positions L0 and L+1 (for nomenclature see ref. 7 and Supplementary Fig. 1b) binds in two adjacent hydrophobic pockets on the small sigma2 subunit that are lined by several hydrophobic residues (Fig. 2), most of which are conserved in all sigma subunits from APs 1–4 in species from yeast to mammals (Fig. 3 and Supplementary Information). Mutation of several of these to hydrophilic residues (sigma2L65S, sigma2V88D, sigma2V98S or sigma2L103S), or filling in the pocket by replacement of sigma2A63 or sigma2N92 with tryptophan, strongly inhibited the binding of recombinant AP2 core complexes to different dileucine motifs displayed on PtdIns(4,5)P2-containing liposomes (Supplementary Table 2, Methods and ref. 12). The Kd for the binding of wild-type AP2 to the CD4 dileucine motif was 0.85 muM, whereas the sigma2V88 and sigma2L103 mutations decreased binding to below detectable levels. A double mutant in sigma2R15 and alphaR21 decreased binding to the dileucine motif by around an order of magnitude, with the Kd for sigma2R15S/sigma2R21E double mutant decreasing to 7.8 muM. In contrast, the binding of wild-type and mutant AP2 cores to PtdIns(4,5)P2 and to YxxPhi was unaffected, with Kd values for binding to the TGN38 YxxPhi motif between 0.31 and 0.38 muM, indicating that all recombinant core variants were correctly folded and functional (Fig. 3).

Figure 2: Details of binding of the CD4 dileucine signal by the sigma2 and alpha subunits of AP2.
Figure 2 : Details of binding of the CD4 dileucine signal by the |[sgr]|2 and |[agr]| subunits of AP2. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

a, The dileucine peptide is bound mainly by the pale blue sigma2 subunit near its interface with the alpha subunit (dark blue). The peptide is shown in its final 2mFo - DFc electron density (cropped around the peptide and contoured at 0.11e Å-3). b, Schematic representation of the dileucine peptide with the principal side chains involved in its binding. Mutants in the boxed residues were analysed kinetically. c, Details of the polar Q(L-4) binding pocket. d, As c, with a semi-transparent electrostatic surface representation (coloured from red at -0.5 V to blue at +0.5 V), showing the positive charge principally due to alphaR21, sigma2K13 and sigma2R15. e, Details of the deep and shallow pockets on sigma2 involved in recognizing the leucine residues at positions L0 and L+1. f, As e, with a molecular surface. Labelled side chains are in the sigma2 subunit unless otherwise indicated.

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Figure 3: Confirmation of location and conservation among different sigma subunits of the dileucine-motif-binding site.
Figure 3 : Confirmation of location and conservation among different |[sgr]| subunits of the dileucine-motif-binding site. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

a, The dileucine-peptide-binding site with residues whose mutation strongly inhibit dileucine peptide binding while not affecting YxxPhi motif binding are coloured red (see Supplementary Table 1 and Supplementary Fig. 8). b, c, Sensorgrams and Kd values for binding of wild-type AP2 core and three mutants thereof that strongly inhibit the binding of AP2 to PtdIns(4,5)P2-containing liposomes displaying the CD4 Q peptide motif (b) but do not affect binding to PtdIns(4,5)P2-containing liposomes displaying the TGN38 YxxPhi motif (c). WT, wild-type; RU, resonance units. d, Mammalian sigma2, sigma1a, sigma3 and sigma4 were aligned by using ClustalW (Supplementary Fig. 7) and the residue conservation was plotted from dark purple (absolute conservation) to white (no conservation) onto the surface of mammalian sigma2 in two views related by a rotation of 180°. The binding site for the dileucine-motif peptide is the outstanding feature of surface residue conservation.

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The L-4 position of AP2-binding dileucine motifs is most commonly occupied by the acidic residues glutamate or aspartate; however, other residues are possible, such as glutamine (as in the human CD4 dileucine motif used here), histidine (in feline CD4) or arginine (in GLUT4) (reviewed in ref. 7). The binding of the residue in the L-4 position occurs on a hydrophilic patch with an overall positive charge. The electron density for its side chain is not as strong as that for the two leucine residues or the peptide backbone, indicating some degree of flexibility in binding. Indeed, simultaneous mutation of two basic residues in the patch (alphaR21 and sigma2R15) were necessary to decrease dileucine motif binding significantly (Fig. 3). In the E peptide, where the L-4 position is the more commonly found glutamate7, the electron density is similar to that for the corresponding glutamine in the Q peptide complex (see Supplementary Information). The use of general electrostatic complementarity rather than a specific interaction between side chains is supported by the fact that exchange of a motif's L-4 glutamate to a shorter aspartate has little effect on the binding of dileucine motifs to AP2 (ref. 11). This general electrostatic interaction probably assists in correctly orienting the peptide, a proposal supported by the observations that mutation of the L-4 acidic residue to alanine significantly weakens binding but does not abolish it, whereas mutation to a positively charged arginine abrogates binding altogether in the context of the sequence EDEPLL11. Consistent with this was our observation of a slightly weaker binding of AP2 to the Q peptide than to the E peptide (see data in Supplementary Table 3). However, in GLUT4 transporters (RRTPSLL) the L-4 and L-5 positions of the internalization motifs are arginine residues (reviewed in ref. 7); suggesting that AP2 might possess alternative modes of dileucine motif binding that would probably use the same dileucine-moiety binding pocket but could place the remainder of the peptide in a different location. Taken together, these structural and mutagenesis data explain the inhibition of the endocytosis of dileucine-motif-containing cargoes when the key L[LI] and [ED] residues are mutated11, 13, 14.

Point mutation and motif swapping indicates that the identity of the residues in the L-1 to L-3 positions have some effect on motif binding10, 11. In the structure presented here, the arginine in the L-1 position interacts with sigma2N97. A proline, often found at this position, would increase the strength of binding by favouring the conformation of the peptide backbone observed in the bound peptide14. In this AP2–CD4 peptide complex the amino-acid residue side chains at the -2 and -3 positions point into solvent, away from the AP2 surface. In vivo, however, the conformational context of the motif may be affected by these and other surrounding residues, as has been proposed for the L-5 position phosphorylated serine residue, which has no visible electron density for the phosphate in the structures presented here, despite its presence as confirmed by mass spectrometry (data not shown). The apparent lack of sequence specificity for residues other than the L[LI] moiety—manifested in the increased tolerance of AP2 for alterations in residues adjacent to the L[LI] in comparison with AP1 and AP3 (ref. 11)—is reflected in the lack of strong contacts between non-L[LI] residues of the motif and AP2 shown here. Such comparatively broad specificity, as is demonstrated by the similar binding affinities displayed by AP2 for several different 'acidic dileucine' motifs (Supplementary Table 3), may ensure the efficient internalization of dileucine-motif-containing proteins that arrive at or mis-sort to the cell surface. The ability of the other AP complexes to discriminate between different L[LI] signals may be necessary to 'fine tune' the directing of cargoes to different intracellular compartments.

In the closed and therefore inactive conformation of AP2 (ref. 3), the hydrophobic L[LI] binding pockets are occupied by side chains from residues in the N-terminal extension to the first helix of the beta2 subunit. beta2F7 occupies the deep LØ pocket, whereas beta2Y6 lies in the broader and shallower L+1 L[LI] pocket (Fig. 4 and Supplementary Fig. 8c). Phosphorylation of beta2Y6 by EGFR15 would inhibit the binding of beta2Y6 in the L+1 pocket and therefore would greatly weaken the interaction between the N terminus of beta2 and sigma2. In the overall structure of the heterotetrameric complex, the helical solenoids of the alpha and beta2 chains form a puckered ring around sigma2 and the N-terminal domain of mu2 (N-mu2), which together make a bowl for the carboxy-terminal domain of mu2 (C-mu2). Comparing this dileucine-motif-liganded, unlocked structure with the 'inactive' conformation, the alpha/beta2 ring splits approximately as two rigid bodies, alpha(1–400)–sigma2 and alpha(401–end) beta2/N–mu2, with a relative rotation of about 20° around a point near residue alpha400. This widens the opening of the ring at the N terminus of beta2, and the N-terminal extension swings away into solvent, thus exposing the peptide-binding site. C-mu2 forms a third rigid body, bridging between alpha and beta2, and roughly preserving its contacts with both alpha and beta2, such that the YxxPhi-binding site on mu2 remains blocked by beta2V365 and beta2Y405. In AP1, and very probably therefore also in AP2, it has been proposed that the binding of either an acidic dileucine or a YxxPhi motif drives a conformational change that favours the binding of the other motif16. A further conformational change must therefore occur so as to produce a fully active conformation that is able to bind both types of motif and PtdIns(4,5)P2 simultaneously. The failure to attain a fully active 'open' conformation is driven by a combination of factors including crystal packing, the lack of a YxxPhi peptide to compete out the autoinhibitory binding of beta2 to mu2, and the high concentration of ammonium sulphate in the crystallization conditions, increasing the apparent strength of the interaction between beta2V365 and the C-mu2 Phi-residue binding pocket and also helping to stabilize the interaction of beta2 residues 350 and 500 with C-mu2.

Figure 4: A conformational change in AP2 is required for dileucine peptide binding.
Figure 4 : A conformational change in AP2 is required for dileucine peptide binding. Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, or to obtain a text description, please contact npg@nature.com

a, In the IP6-liganded inactive conformation the N terminus of beta2 is held in place by beta2F7 and beta2Y6 sitting in the L0 and L+1 pockets. b, For dileucine motifs to bind, the N terminus of beta2 is displaced from the surface of sigma2. c, Close-up of the dileucine-binding site: the dileucine peptide (gold) runs in the opposite direction to the N terminus of beta2 (green). d, Schematic representation of the conformational change: a 20° hinge movement in alpha moves the N terminus of beta2 out of the dileucine-binding site, allowing the motif to bind. The mu2 subunit has been omitted for clarity. e, Front and back views of the conformational change: the IP6-liganded inactive conformation is shown in pale colours, the unlocked dileucine-peptide-bound conformation in dark colours. The YxxPhi site on mu2 remains blocked, and is remote from the dileucine-binding site.

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The binding site on sigma2 used by AP2 to bind acidic dileucine motifs is not the same as that used by other proteins with sigma-like or 'longin domain' folds to bind their cognate ligands: these include the SNAREs Sec22b17 and Vamp7 (ref. 5) and the signal recognition particle SRalpha subunit18. However, the hydrophobic L[LI]-binding pocket, but not the acidic-residue-binding patch at the -4 position, is largely conserved in the zetaCOP subunit of the betagammadeltazeta subcomplex of COPI that is homologous in structure to AP complexes (PDB 2HF6 and Supplementary Information), pointing to a possible conservation of function in the binding of a di-hydrophobic motif, candidates for which would include the FF motif of the COPI cargoes, the p24 family and ERGIC-53 (ref. 19) and the N terminus of beta-COP.

The mechanism of the binding of a dileucine motif by the AP2 clathrin adaptor complex conforms to the established model of a CCV–cargo interaction. The peptide is bound in an extended conformation by a folded adaptor domain, with specificity arising from a few residue side chains fitting into compatible pockets. The mode of binding is therefore superficially similar to the binding of DxxLL motifs by the VHS domains of GGAs8, 9, but the design of the LL and acidic-residue recognition pockets, their spacing and the structures underlying them are completely different (Supplementary Fig. 9). The interaction between the two isolated components, a dileucine-motif-containing peptide molecule and a single AP2 complex, is dynamic, with a Kd in the micromolar range (1–3 muM)12, and buries about 1,100 Å2 of solvent-accessible surface area. These values are both similar to those shown for other cargo–clathrin adaptor interactions6. When the signal is presented in, and so simultaneously recognized with, a PtdIns(4,5)P2 -containing membrane, the apparent strength of the interaction with AP2 is increased: the Kd decreases to the high nanomolar range, as shown here, and it is this coincident detection of PtdIns(4,5)P2 and acidic dileucine motif that allows the signal to outcompete the N terminus of beta2 for binding to sigma2. The need for sorting signals to be presented in a PtdIns(4,5)P2-containing membrane for efficient AP2 binding also prevents inappropriate recognition of similar sequences in cytoplasmic proteins.

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Methods Summary

Recombinant AP2 cores were expressed and purified as in ref. 3. Crystals of the CD4–AP2-core complexes were grown over a period of three weeks by hanging-drop vapour diffusion against a reservoir containing 1.7–2.2 M ammonium sulphate, sodium citrate pH 6.5 and 5 mM dithiothreitol from a mixture of 10 mg ml-1 AP2 core and 7 mg ml-1 peptide. Crystals were of space group P43212, with two molecules in the asymmetric unit, and diffracted to a best resolution of 3.0 Å. After cryoprotection, data were collected at 100 K on Diamond beamline I03 and the structure was solved by molecular replacement with PHASER20. A complete explanation of the structure determination and of work performed to confirm the identity and orientation of the peptide is given in Methods. Mutant versions of AP2 core were sequenced throughout and the surface plasmon resonance (SPR)-based liposome binding assay was performed as in ref. 12, with the peptides described in Methods.

Full methods accompany this paper.

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References

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Supplementary Information

Supplementary information accompanies this paper.

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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).

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Author Information

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.

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Online Methods

Expression, crystallization and structure solution

The four subunits of the AP2 core (1–621 mouse alpha-adaptin; 1–591 human beta2-adaptin; 1–435 rat mu2-adaptin, 1–143 mouse sigma2-adaptin) were co-expressed in Escherichia coli from two bicistronic plasmids of different (ampicillin and kanamycin) antibiotic resistances as in ref. 3 in E. coli BL21DE3 pLysS cells at 22 °C. Protein expression was allowed to continue for 14 h; the cells were harvested and lysed with a Cell Disrupter (Constant Cell Disruption Systems), and insoluble material was removed by centrifugation. AP2 was purified throughout in 10 mM Tris pH 8.7, 250 mM NaCl, 1 mM dithiothreitol (DTT). The complex was bound to glutathione-Sepharose (Pharmacia), the column was extensively washed and the complex was eluted by cleavage overnight at 20 °C with bovine thrombin (Sigma). The resulting AP2 complex was bound to Ni2+-nitrilotriacetate-agarose (Qiagen), the column was extensively washed and the complex was eluted with buffer containing 0.3 M imidazole. The complex was then purified by gel filtration on Superdex S200 (Pharmacia) and finally passed back down a glutathione-Sepharose column to remove any remaining uncleaved glutathione S-transferase (GST)–AP2 complex. The yield was 1–2 mg of purified AP2 per litre of culture.

Crystals of the CD4 peptide/AP2 core complexes were grown over a period of three weeks by hanging-drop vapour diffusion against a reservoir containing 1.7–2.2 M ammonium sulphate, 100 mM sodium citrate pH 6.5 and 5 mM DTT from a mixture of 10 mg ml-1 AP2 core and 7 mg ml-1 peptide. The best crystals had dimensions of about 200 mum and diffracted to a best resolution of 3.0 Å. Crystals were cryoprotected with 1.8–2.3 M ammonium sulphate, 100 mM sodium citrate pH 6.5, 17% glycerol and 7 mg ml-1 CD4 dileucine peptide. Data were collected at 100 K on Diamond beamline I03, integrated with Mosflm21 and scaled with Scala22. The crystals belong to space group P43212, with two molecules in the asymmetric unit. The structure was solved by molecular replacement by using Phaser20, with models derived from the IP6-liganded 'inactive' AP2 core structure (ref. 3; PDB code 2VGL). Extra electron density representing the peptide bound to the sigma2 subunit was clear from the first maps after molecular replacement (Supplementary Fig. 1). The final model refined at 3.0 Å resolution comprises residues 3–623 of alpha (lacking 1–2 and including a final two residues from the cloning linker), 12–582 of beta2 (lacking 1–11 and 583–591), 1–142 of sigma2 (lacking none), and residues 1–141 and 159–435 of mu2 (lacking 142–158, as well as the loop 223–231, which are also missing from the structure of isolated C-mu2), the dileucine peptide, sulphates and waters. For statistics relating to the data collection and structure solution see Supplementary Table 1 and for a sample of typical electron density see Supplementary Fig. 2.

The orientation and registration of the RMS(P)QIKRLLSE peptide in this density was decided as follows. First, there were clearly two pockets for hydrophobic residues, one deep and one shallow. Electron density consistent with leucine side chains could be seen in each pocket, but the orientation of the backbone connecting them could be in either direction. Second, the electron density for the peptide extends for about four or five residues on one side of the deep pocket, but not more than one or two on the other side of the shallow pocket; because the LL sequence is only two residues from the C terminus of the peptide, the longer part of the density must be N-terminal to the LL sequence. This places the invariant first L in the deep pocket, and the conserved second L (which can also be I or M) in the shallower pocket. With this model of the peptide, the structure refined well, showing good density for the residues SQIKRLLS (see Fig. 2), but no density for the phosphate moiety of the phosphoserine (S(P)) side chain, which was therefore modelled as a serine, although mass spectrometry of a sample of peptide incubated in crystallization conditions for two weeks confirmed that the serine residue remained phosphorylated. The lack of density for the phosphate is likely to be caused by static disorder of the phosphate group. The structure was refined with Phenix.refine23, with default tight non-crystallographic symmetry restraints between the two molecules, and rebuilt with Coot24. Large positive difference density peaks close to Arg or Lys side chains were interpreted as SO42- ions, given the crystallization conditions in 2 M (NH4)2SO4, and a few well-resolved waters were added to the model (Supplementary Fig. 3). TLS (translation-libration-screw) group assignment used the TLSMD server (http://skuld.bmsc.washington.edu/approxtlsmd/)25 splitting the alpha chain into six groups, the beta2 chain into five groups, the mu2 chain into two groups and the sigma2 chain as one group. Refinement without TLS gave slightly higher R factors (Rwork = 0.222, Rfree = 0.266) but little difference in the maps (map correlation 0.99, 0.93 at 3 Å resolution). Ramachandran analysis of the final model showed 86% of residues in the most favoured region, 96% in 'allowed' regions.

The structure of a complex between the AP2 core and a version of the CD4 acidic dileucine motif peptide termed 'E-peptide' in which the L-4 Q residue was replaced with an E (that is, RMSpEIKRLLSE) was also solved. The 'E-peptide' data set was of lower quality than the best Q-peptide data set. The crystal used to collect the Q-peptide data set gave better diffraction than the many others that were tried, and also had the serendipitous advantage of rotating approximately around the long c axis, which reduced the spot overlap compared with rotation around an axis roughly perpendicular to the long axis, as for the E-peptide crystal. The final model from the Q-peptide after the Q to E substitution was refined against the E-peptide data, with and without the peptide; as expected, the map quality was poorer than for the Q-peptide, but there was no significant difference in the structure. The electron density for the peptide shows no evidence of any difference in conformation (Supplementary Fig. 4), nor are the residues contacting the E side chain noticeably different: in particular, the side-chain density for alphaR21 is still very weak. The lack of crystallizability of other AP2 core:dileucine motif peptide complexes is likely to be explained by the packing interaction that occur in the crystal between the L-1 position arginine in the peptide and the side chain of beta2Glu157 and the backbone amide of beta2Q154.

Peptide sequences, conjugation of peptides to lipid and liposome and analyte preparation

The following peptides were coupled to lipid for incorporation into liposomes, which were then used in the SPR-based binding experiments: TGN38*, CKVTRRPKASDYQRL; phosphorylated CD4 Q peptide, CHRRRQAERM(SP)QIKRLLSEK; phosphorylated CD4 E peptide, CHRRRQAERM(SP)EIKRLLSEK; limp-II, CRGQGSTDEGTADERAPLIRT; tyrosinase*, CKKQPQEERQPLLMDKDDYHSLLYQSHL; TRP1*, CRSRSTKNEANQPLLTDHYQRYAED; LRP*, CKVTRRPKASEDEPLLS.

Peptides with an asterisk were also synthesized with mutations in their critical sorting-relevant amino-acid residues (Yright arrowA in TGN38 and pairs of alanine residues substituting for the two leucine residues in dileucine-containing peptides) and served as negative controls.

Coupling of peptides to 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(4-(p-maleimidophenyl) butyramide) (MPB-PE) and the subsequent incorporation into liposomes were performed as described in ref. 12. In brief, peptides were linked covalently through their N-terminal cysteine to MPB-PE (Avanti). The lipid-linked peptide was subsequently incorporated into 100 nm liposomes that were composed of 60% phosphatidylcholine (PC), 20% phosphatidylethanolamine (PE), 10% PtdIns(4,5)P2 and 10% peptido-lipid. The efficiency of peptide coupling was determined by mass spectrometry, and the integrity of the liposomes was controlled by dynamic light scattering. The average variation of the PtdIns(4,5)P2 content between different liposome preparations was less than 3% as determined by binding of the ENTH domain of epsin1 (see Supplementary Fig. 5).

Peptido-liposomes were used to generate a stable 'membrane mimic' on an L1 sensor surface of a BIAcore 3000 SPR biosensor (BIAcore AB). The system was first equilibrated in 10 mM Tris pH 8.7, 250 mM NaCl, 1 mM DTT, which was used as the running buffer, followed by priming with two injections of 20 mM CHAPS for 1 min (flow rate 10 mul min-1) and injection of the liposomes (0.25 mM final concentration) for 4 min at 5 mul min-1. Loosely bound liposomes were removed by two pulse injections of 50 mM sodium hydroxide for 30 s at 30 mul min-1. This procedure resulted in an increase of the baseline by about 9500 resonance units (RU) with less than 4% variation between the four flow cells. After liposome capture, wild-type and mutant AP2 cores were injected at concentrations ranging from 50 nM to 1 muM at a flow rate of 30 mul min-1 for 1 min (association) followed by buffer flow for 4 min (dissociation). All protein that did not dissociate within this period from the membrane was stripped off by a 20-s pulse injection of 50 mM NaOH. In a typical experiment the sensor surface was derivatized with basic liposomes (PC/PE) in flow cell 1, PC/PE plus PtdIns(4,5)P2 in flow cell 2, PC/PE plus PtdIns(4,5)P2 plus TGN38 peptide in flow cell 3, and PC/PE plus PtdIns(4,5)P2 plus phosphoCD4 peptide in flow cell 4. This setup allowed us to differentiate between background binding to PC/PE liposomes, binding to PtdIns(4,5)P2 and binding to PtdIns(4,5)P2 together with a sorting-signal-containing peptide. In other experiments, TGN38 and LRP dileucine sorting signal containing peptido-liposomes were compared with liposomes containing mutant versions of both peptides in which the critical tyrosine or both leucine residues had been replaced with alanine. This setup was used to control binding of AP2 to a mutated sorting signal in a membrane containing PtdIns(4,5)P2 (not shown). The kinetic analysis of binding was performed exactly as described26 and rate constants were calculated with the evaluation software supplied by the manufacturer (BIAcore evaluation software27). All GST–AP2 complexes used in the SPR binding assay were expressed to similar levels and were checked for intactness and folding by SDS–PAGE and circular dichroism before use. A typical Coomassie-blue-stained SDS–PAGE gel of AP2 cores used in the SPR-based assay is shown in Supplementary Fig. 5. The samples were actually those used in the determination of Kd values shown in Fig. 3 in the main text.

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