the four subunits of the ap2 core ( 1 - 621 mouse -adaptin ; 1 - 591 human 2-adaptin ; 1 - 435 rat 2-adaptin , 1 - 143 mouse 2-adaptin ) were co - expressed in e. coli from two bicistronic plasmids of different ( ampicillin and kanamycin ) antibiotic resistances as in   in e. coli bl21de3 plyss cells at 22c .
protein expression was allowed to continue for 14 hours , the cells harvested , lysed using a cell disrupter ( constant cell disruption systems ) and insoluble material removed by centrifugation .
ap2 was purified throughout in 10 mm tris ph 8.7 , 250mmnacl , 1 mm dtt .
the complex was bound to glutathione sepharose ( pharmacia ) , the column extensively washed and the complex eluted by overnight cleavage at 20c with bovine thrombin ( sigma ) .
the resulting ap2 complex was bound to ninta agarose ( quiagen ) , the column extensively washed and the complex 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 gst - ap2 complex .
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 ph6.5 and 5 mm dtt from a mixture of 10mg / ml ap2 core and 7mg / ml peptide .
the best crystals had dimensions of around 200m and diffracted to a best resolution of 3.0 .
crystals were cryoprotected with 1.8 - 2.3 m ammonium sulphate , 100 mm sodium citrate ph 6.5 , 17% glycerol and 7mg / ml cd4 dileucine peptide .
data were collected at 100k on diamond beamline i03 , integrated using mosflm , scaled with scala .
the structure was solved by molecular replacement using phaser , with models derived from the ip6-liganded 
extra electron density representing the peptide bound to the 2 subunit was clear from the first maps after molecular replacement ( figure s1 ) .
the final model refined at 3.0 resolution comprises residues 3 - 623 of  ( missing 1 - 2 and including a final 2 residues from the cloning linker ) , 12 - 582 of 2 ( missing 1 - 11 and 583 - 591 ) , 1 - 142 of 2 ( missing none ) , and residues 1 - 141 and 159 - 435 of 2 ( missing 142 - 158 , as well as the loop 223 - 231 which are also missing in the structure of isolated c-2 ) , the dileucine peptide , sulphates and waters . for statistics relating to the data collection and structure solution see table 1 and for a sample of typical electron density see figures2 .
the orientation and registration of the rms(p)qikrllse peptide in this density was decided as follows .
( 1 ) 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 .
( 2 ) the electron density for the peptide extends for about 4 or 5 residues on one side of the deep pocket , but not more than 1 or 2 on the other side of the shallow pocket : since 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 figure 2 ) , but no density for the phosphate moiety of the phophoserine ( s(p ) ) side chain , which was therefore modelled as a serine , although mass spectrometry of a sample of peptide incubated in crystallisation conditions for 2 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 using phenix.refine   with default tight ncs restraints between the two molecules , and rebuilt with coot .
large positive difference density peaks close to arg or lys side chains were interpreted as so4 ions , given the crystallisation conditions in 2 m ( nh4)2so4 , and a few well resolved waters were added to the model ( figure s3 ) .
tls group assignment used the tlsmd server ( http://skuld.bmsc.washington.edu/~tlsmd/ )   splitting the  chain into 6 groups , the 2 chain into 5 , the 2 chain into 2 groups and the 2 chain as 1 group .
refinement without tls gave slightly higher rfactors ( rwork 0.222 , rfree 0.266 ) but little difference in the maps ( map correlation 0.99 , 0.93 at 3 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 ( ie rmspeikrllse ) was also solved .
the crystal used to collect the q - peptide dataset gave better diffraction than the many others which were tried , and also had the serendipitous advantage of rotating approximately around the long c axis , which reduced the spot overlap compared to 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 & 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 ( figure s4 ) , nor are the residues contacting the e side chain noticeably different : in particular , the side chain density for r21 is still very weak . the lack of crystallisability of other ap2 core : dileucine motif peptide complexes is likely 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 2glu157 and the backbone amide of 2q154 the following peptides were coupled to lipid for incorporation into liposomes , which were then used in the spr - based binding experiments : 
 tgn 38*ckvtrrpkasdyqrl , phosphorylated cd4 q peptidechrrrqaerm(sp)qikrllsekphosphorylated cd4 e peptidechrrrqaerm(sp)eikrllseklimp - iicrgqgstdegtaderaplirttyrosinase*ckkqpqeerqpllmdkddyhsllyqshltrp1*crsrstkneanqplltdhyqryaedlrp*ckvtrrpkasedeplls peptides with and asterisk were also synthesized with mutations in their critical sorting relevant amino acid residues ( y to a in tgn 38 and pairs of alanine residues substituting for the two leucine residues in dileucine containing peptides ) and served as negative controls .
coupling of peptides to mpb - pe and the subsequent incorporation into liposomes were performed as described in . in brief , peptides were covalently linked via their amino - terminal cysteine to 1,2-dipalmitoyl - sn - glycero-3-phosphoethanolamine - n-[4-(p - maleimidophenyl ) butyramide ] , avanti , usa . the lipid - linked peptide was subsequently incorporated into 100 nm liposomes that were composed of 60% pc , 20%pe , 10% ptdins4,52 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 ptdins4,5p2 content between different liposome preparations was less than 3% as determined by binding of the enth domain of epsin1 ( see figure s5 ) .
peptido - liposomes were used to generate a stable  membrane mimic  on a l1sensorsurface of a biacore 3000 ( biacore ab , sweden ) spr biosensor .
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 1min ( flow rate 10l / min ) and injection of the liposomes ( 0.25 mm final concentration ) for 4min at 5l / min .
loosely bound liposomes were removed by two pulse injections of 50 mm sodium hydroxide for 30sec at 30l / min .
after liposome capture , wild type and mutant ap2 cores were injected at concentrations ranging from 50 nm to 1m at a flow rate of 30l / min for one 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 20sec 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+ ptdins4,5p2 in flow cell 2 , pc / pe+ ptdins4,5p2 + tgn38 peptide in flow - cell 3 and with pc / pe+ ptdins4,5p2 + phosphocd4 peptide in flow - cell 4 .
this set up allowed to differentiate between background binding to pc / pe liposomes , binding to ptdins4,5p2 and ptdins4,5p2 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 were substituted for alanine .
this setup was used to control binding of ap2 to a mutated sorting signal in a ptdins4,5p2 containing membrane ( not shown ) .
the kinetic analysis of binding was carried out exactly as described   and rate constants were calculated using the evaluation software supplied by the manufacturer ( biacore evaluation software ) .
all gst - ap2 complexes used in the spr binding assay expressed to similar levels and were checked for intactness and folding by sds page and circular dichroism prior to use .
a typical coomassie blue stained sds page gel of ap2 cores used in the spr - based assay is shown in figure s5 .
the samples are actually those used in the determination of kds shown in figure 3 in the main text .
the four subunits of the ap2 core ( 1 - 621 mouse -adaptin ; 1 - 591 human 2-adaptin ; 1 - 435 rat 2-adaptin , 1 - 143 mouse 2-adaptin ) were co - expressed in e. coli from two bicistronic plasmids of different ( ampicillin and kanamycin ) antibiotic resistances as in   in e. coli bl21de3 plyss cells at 22c .
protein expression was allowed to continue for 14 hours , the cells harvested , lysed using a cell disrupter ( constant cell disruption systems ) and insoluble material removed by centrifugation .
ap2 was purified throughout in 10 mm tris ph 8.7 , 250mmnacl , 1 mm dtt .
the complex was bound to glutathione sepharose ( pharmacia ) , the column extensively washed and the complex eluted by overnight cleavage at 20c with bovine thrombin ( sigma ) .
the resulting ap2 complex was bound to ninta agarose ( quiagen ) , the column extensively washed and the complex 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 gst - ap2 complex .
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 ph6.5 and 5 mm dtt from a mixture of 10mg / ml ap2 core and 7mg / ml peptide .
the best crystals had dimensions of around 200m and diffracted to a best resolution of 3.0 .
crystals were cryoprotected with 1.8 - 2.3 m ammonium sulphate , 100 mm sodium citrate ph 6.5 , 17% glycerol and 7mg / ml cd4 dileucine peptide .
data were collected at 100k on diamond beamline i03 , integrated using mosflm , scaled with scala .
the structure was solved by molecular replacement using phaser , with models derived from the ip6-liganded 
extra electron density representing the peptide bound to the 2 subunit was clear from the first maps after molecular replacement ( figure s1 ) .
the final model refined at 3.0 resolution comprises residues 3 - 623 of  ( missing 1 - 2 and including a final 2 residues from the cloning linker ) , 12 - 582 of 2 ( missing 1 - 11 and 583 - 591 ) , 1 - 142 of 2 ( missing none ) , and residues 1 - 141 and 159 - 435 of 2 ( missing 142 - 158 , as well as the loop 223 - 231 which are also missing in the structure of isolated c-2 ) , the dileucine peptide , sulphates and waters . for statistics relating to the data collection and structure solution see table 1 and for a sample of typical electron density see figures2 .
the orientation and registration of the rms(p)qikrllse peptide in this density was decided as follows .
( 1 ) 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 .
( 2 ) the electron density for the peptide extends for about 4 or 5 residues on one side of the deep pocket , but not more than 1 or 2 on the other side of the shallow pocket : since 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 figure 2 ) , but no density for the phosphate moiety of the phophoserine ( s(p ) ) side chain , which was therefore modelled as a serine , although mass spectrometry of a sample of peptide incubated in crystallisation conditions for 2 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 using phenix.refine   with default tight ncs restraints between the two molecules , and rebuilt with coot .
large positive difference density peaks close to arg or lys side chains were interpreted as so4 ions , given the crystallisation conditions in 2 m ( nh4)2so4 , and a few well resolved waters were added to the model ( figure s3 ) .
tls group assignment used the tlsmd server ( http://skuld.bmsc.washington.edu/~tlsmd/ )   splitting the  chain into 6 groups , the 2 chain into 5 , the 2 chain into 2 groups and the 2 chain as 1 group .
refinement without tls gave slightly higher rfactors ( rwork 0.222 , rfree 0.266 ) but little difference in the maps ( map correlation 0.99 , 0.93 at 3 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 ( ie rmspeikrllse ) was also solved .
the crystal used to collect the q - peptide dataset gave better diffraction than the many others which were tried , and also had the serendipitous advantage of rotating approximately around the long c axis , which reduced the spot overlap compared to 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 & 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 ( figure s4 ) , nor are the residues contacting the e side chain noticeably different : in particular , the side chain density for r21 is still very weak . the lack of crystallisability of other ap2 core : dileucine motif peptide complexes is likely 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 2glu157 and the backbone amide of 2q154
the following peptides were coupled to lipid for incorporation into liposomes , which were then used in the spr - based binding experiments : 
 tgn 38*ckvtrrpkasdyqrl , phosphorylated cd4 q peptidechrrrqaerm(sp)qikrllsekphosphorylated cd4 e peptidechrrrqaerm(sp)eikrllseklimp - iicrgqgstdegtaderaplirttyrosinase*ckkqpqeerqpllmdkddyhsllyqshltrp1*crsrstkneanqplltdhyqryaedlrp*ckvtrrpkasedeplls peptides with and asterisk were also synthesized with mutations in their critical sorting relevant amino acid residues ( y to a in tgn 38 and pairs of alanine residues substituting for the two leucine residues in dileucine containing peptides ) and served as negative controls .
coupling of peptides to mpb - pe and the subsequent incorporation into liposomes were performed as described in . in brief , peptides were covalently linked via their amino - terminal cysteine to 1,2-dipalmitoyl - sn - glycero-3-phosphoethanolamine - n-[4-(p - maleimidophenyl ) butyramide ] , avanti , usa . the lipid - linked peptide was subsequently incorporated into 100 nm liposomes that were composed of 60% pc , 20%pe , 10% ptdins4,52 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 ptdins4,5p2 content between different liposome preparations was less than 3% as determined by binding of the enth domain of epsin1 ( see figure s5 ) .
peptido - liposomes were used to generate a stable  membrane mimic  on a l1sensorsurface of a biacore 3000 ( biacore ab , sweden ) spr biosensor .
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 1min ( flow rate 10l / min ) and injection of the liposomes ( 0.25 mm final concentration ) for 4min at 5l / min .
loosely bound liposomes were removed by two pulse injections of 50 mm sodium hydroxide for 30sec at 30l / min .
after liposome capture , wild type and mutant ap2 cores were injected at concentrations ranging from 50 nm to 1m at a flow rate of 30l / min for one 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 20sec 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+ ptdins4,5p2 in flow cell 2 , pc / pe+ ptdins4,5p2 + tgn38 peptide in flow - cell 3 and with pc / pe+ ptdins4,5p2 + phosphocd4 peptide in flow - cell 4 .
this set up allowed to differentiate between background binding to pc / pe liposomes , binding to ptdins4,5p2 and ptdins4,5p2 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 were substituted for alanine . this setup was used to control binding of ap2 to a mutated sorting signal in a ptdins4,5p2 containing membrane ( not shown ) .
the kinetic analysis of binding was carried out exactly as described   and rate constants were calculated using the evaluation software supplied by the manufacturer ( biacore evaluation software ) .
all gst - ap2 complexes used in the spr binding assay expressed to similar levels and were checked for intactness and folding by sds page and circular dichroism prior to use .
a typical coomassie blue stained sds page gel of ap2 cores used in the spr - based assay is shown in figure s5 .
the samples are actually those used in the determination of kds shown in figure 3 in the main text .
 summarymost 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 . in the case of clathrin - coated vesicles ( ccvs ) 
 the motifs are recognised by clathrin adaptors . 
 the ap2 adaptor complex ( subunits ,2,2,2 ) recognises both major endocytic motifs : yxx motifs 1 and [ de]xxxl[li ] acidic dileucine motifs . here 
 we describe the binding of ap2 to the endocytic dileucine motif from cd4 2 . 
 the major recognition events are the two leucine residues binding in hydrophobic pockets on 2 . 
 the hydrophilic residue four residues upstream from the first leucine sits on a positively charged patch made from residues on 2 and  subunits . 
 mutations in key residues inhibit the binding of ap2 to  
 acidic dileucine motifs displayed in liposomes containing ptdins4,5p2 , but do not affect binding to yxx motifs via 2 . in the  inactive ap2 core structure 3 , both motif binding sites are blocked by different parts of the 2 subunit . to allow a dileucine motif to bind , the 2 n - terminus is displaced and becomes disordered ; however , in this structure the yxx binding site on 2 remains blocked . 
Online Materials and Methods
Expression, crystallisation & structure solution
Peptides sequences, conjugation of peptides to lipid and liposome and analyte preparation
Supplementary Material