the peptides fdnpvyqktt , fdnpvy , fdnpvsqktt , fdnpvs ,   fdnpva , fdnpvf , and fdnpvl were synthesized using standard   fmoc chemistry with a free cooh terminus and an acylated nh2 terminus .
crude peptides were purified by reverse - phase hplc on a c18 column ( vydac , hesperia , ca ) , equilibrated in h2o with 0.1% trifluoroacetic acid , and eluted with a linear gradient of acetonitrile .
the identities of   the peptides were confirmed by mass spectrometry and amino acid analysis . concentrated stock solutions of peptides
were made by dissolving the   peptide in d2o and adjusting the ph to 6.0 with aliquots of concentrated   naoh .
the coat proteins were then gel - filtered over a superdex 200   column ( pharmacia biotech , inc . ,
clathrin and ap fractions were pooled and loaded directly   onto an anion exchange column ( source q 15 ; pharmacia biotech , inc . )  
equilibrated in 20 mm ethanolamine , ph 9.0 , and eluted with a steep linear gradient of 1 m nabr .
clathrin and ap fractions were pooled and concentrated using centriprep 30 ( amicon corp . ,
easton , tx ) . concentrated   proteins were polymerized ( 25 ) by extensive dialysis against nmr sample   buffer ( 40 mm d4-malonic acid [ cambridge isotope labs , andover , ma ] ,   ph 6.0 , 0.2 mm edta , 0.2 mm dtt , and 3 mm nan3 ) .
protein concentration of the polymerized cage complex was determined using the bradford reagent ( bio - rad laboratories , hercules , ca ) . for preparation of cages composed of clathrin or clathrin and ap2 ,
an additional anion exchange chromatography step was performed to separate clathrin from ap2 before gel filtration .
ap-2 containing fractions from the second anion exchange step were then pooled   on the basis of coomassie blue  stained sds - page .
the clathrin fraction   was divided in half , and all of the ap-2 sample was added to one half of   the clathrin .
equal total protein amounts of   clathrin or clathrin / ap-2 cages were added to each nmr sample , which   contained clathrin ( 9.7 mg / ml ) or clathrin ( 8.5 mg / ml ) and ap-2 ( 1.2 mg/ ml ) .
sample purity was confirmed by coomassie blue  stained sds - page   gels and by western blotting with an antibody against -adaptin ( 100/1 ;   sigma chemical co. , st .
the clathrin terminal domain ( residues 1579 ) was expressed as a glutathione - s - transferase ( gst ) fusion protein in escherichia coli bl21 cells ,   and was prepared as described ( 17 ) .
glutathione agarose beads ( sigma   chemical co. ) with the bound gst construct were then washed with   thrombin cleavage buffer ( 50 mm tris , 2.5 mm cacl2 , 150 mm nacl , ph   7.4 ) .
the beads were incubated overnight with gentle agitation in the presence of thrombin ( sigma chemcial co. ) .
dtt was added to 10 mm and   allowed to incubate an additional 1 h before eluting the cleaved terminal   domain construct from the beads .
the clathrin hub construct ( residues 10741483 ) was expressed as a   hexahistidine construct in e. coli bl21(de ) cells as described ( 29 ) .
bacterial lysates were incubated with ni - nta agarose ( qiagen inc . , valencia , ca ) overnight and eluted with 250 mm imidazole in 50 mm tris , ph   7.8 .
the eluted polypeptides were then loaded directly onto an anion exchange column equilibrated in 30 mm tris , 1 mm edta , ph 7.8 , and   eluted with a linear gradient of 0.5 m kcl .
fractions were pooled on the   basis of coomassie blue  stained sds - page gels .
because the hub   polypeptide aggregated in the nmr sample buffer , both the terminal domain and hub polypeptides were dialyzed against 40 mm kpo4 , 150 mm   kcl , 0.2 mm edta , 3 mm nan3 , and 0.2 mm dtt ph 7.2 .
all spectra were acquired at 500 mhz and 25c in sample buffer containing 10% d2o on a varian unity 500 spectrometer with a 5999.7-hz spectral width using 3-(tetramethylsilyl ) propionic acid as a chemical shift reference .
1d spectra were acquired with 128 transients of 64,000 points zero   filled to 131,072 points ( see fig .
1 ) or 64 transients of 32,000 points zero   filled to 65,536 points ( see figs . 4 and 5 ) .
line - broadening was measured   as the difference of peak widths at half height using the felix 2.30 ( biosym   technologies ) program .
the broadening is reported as the line - width of   the resonance peak in the presence of cages minus the line - width in the   absence of cages .
crosspeak intensities were calculated in arbitrary units   of volume using the felix 2.30 program from spectra acquired at 60- , 100- ,   140- , and 220-ms mixing times .
the spectra were acquired in phase - sensitive mode ( 48 ) with 2  256 fids of 1024 complex points each . a spectral   width of 5999.7 hz
the relative kd for wild - type and position 807 mutant peptides from   clathrin terminal domains was obtained from measurements of the peak   intensities of the phe ( 3 , 5 ) aromatic protons for each of the peptides in   the presence and absence of protein .
the free and bound populations   were calculated from the following equation : \documentclass[10pt]{article } 
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 \begin{equation*}\frac{1}{i_{o}}=\frac{p^{f}}{i_{f}}+\frac{p^{b}}{i_{b}}\end{equation*}\end{document }   where i is the intensity of the free ( if ) , the bound ( ib ) , and observed ( io )   resonance , and p and p are the free and bound populations , respectively .  
since peak intensities are inversely proportional to the transverse relaxation rate ( 1/t2 ) , the population of the free and bound peptide can be estimated from the change in intensity of a given peptide resonance upon addition of clathrin terminal domain if the t2 of the resonance in the free   and bound forms is known .
the t2 for a protein is approximately proportional to the molecular weight , and from the line - widths observed in 20-kd   proteins ( 3 ) , the expected t2 for a 55-kd protein is 10 ms .
the t2 calculated from the line - widths of several resonances of the free peptides was   100 ms .
the values of ib were estimated to be if/10 , assuming that the t2s   of the free and bound resonances are 100 and 10 ms , respectively .
therefore , the calculated populations of p and p were then used to obtain kds   assuming standard protein  ligand equilibrium .
since the kds were based   on t2 estimates , their absolute values are likely to be inaccurate .
however , the relative values of the kds can be compared because they were all   obtained by the same method .
distances were calculated only for interresidue noes seen in 60 ms   mixing time data sets .
sequential h to nh noes were not used because   they contained contributions from the free peptide .
noe distance restraints were calculated from both wild - type hexa- and decapeptide either   by using bins of weak ( w ; 1.82.3  ) , medium ( m ; 1.83.5  ) , and strong   ( s ; 1.85.0  ) peaks , or by measured noe volumes calibrated individually   for each dimension with the average volume of the phe , tyr , and pro   ( / ) noes corresponding to 1.75  .
structures were calculated by simulated annealing performed with the programs insightii and nmrchitect ( biosym technologies , san diego , ca ) incorporating either type   of restraint . in short
, peptides were heated to 1,000 k , and then the distance and chiral restraints were slowly increased over 30 ps .
the peptides   were then slowly cooled to 300 k while increasing covalent and nonbond   forces over 32 ps .
the final structures were then obtained by iteratively   minimizing to a final derivative of 0.01 kcal / mol /  and simulating 30   ps of restrained dynamics .
removal of pseudoatom   restraints for tyr (  ,  ) and phe( ) was based on the geometry of an initial   set of structures . the final set of 30 restraints was ( * denotes pseudoatom   and distance is denoted as s , m , or w ) : ac(me*)::f(h1 ) , w ; f(h1 ) : : d(hn ) , w ; f(h2)::d(hn ) , w ; d(h2)::n(hn ) , w ; n(h1)::p(hs ) , w ;   n(h2)::p(hs ) , m ; n(h1)::p(hr ) , w ; n(h2)::p(hr ) , w ; n(hn ) : : p(hr ) , w ; p(hr)::v(hn ) , m ; p(hs)::v(hn ) , w ; p(hs)::v(hn ) , w ;   v(hn)::y(hn ) , s ; v(hn)::y(h1 ) , w ; v(h1*)::y(h1 ) , w ; v(h1 * ) : : y(h1 ) , w ; ac(me*)::d(hn ) , w ; ac(me*)::n(h21 ) , w ; ac(me * ) : : n(h22 ) , w ; v(hn)::n(h1 ) , w ; v(hn)::n(h2 ) , w ; n(h1)::y(h2 ) , w ;   n(h2)::y(h1 ) , w ; p(hs)::y(h1 ) , w ; p(hs)::y(h1 ) , w ;
p(hs ) : : y(hn ) , m ; p(hr)::y(hn ) , w ; p(hr)::y(h1 ) , w ; p(hr)::y(h1 ) , w ;  
the peptides fdnpvyqktt , fdnpvy , fdnpvsqktt , fdnpvs ,   fdnpva , fdnpvf , and fdnpvl were synthesized using standard   fmoc chemistry with a free cooh terminus and an acylated nh2 terminus .
crude peptides were purified by reverse - phase hplc on a c18 column ( vydac , hesperia , ca ) , equilibrated in h2o with 0.1% trifluoroacetic acid , and eluted with a linear gradient of acetonitrile .
the identities of   the peptides were confirmed by mass spectrometry and amino acid analysis . concentrated stock solutions of peptides
were made by dissolving the   peptide in d2o and adjusting the ph to 6.0 with aliquots of concentrated   naoh .
the coat proteins were then gel - filtered over a superdex 200   column ( pharmacia biotech , inc . ,
clathrin and ap fractions were pooled and loaded directly   onto an anion exchange column ( source q 15 ; pharmacia biotech , inc . )  
equilibrated in 20 mm ethanolamine , ph 9.0 , and eluted with a steep linear gradient of 1 m nabr .
clathrin and ap fractions were pooled and concentrated using centriprep 30 ( amicon corp . ,
easton , tx ) . concentrated   proteins were polymerized ( 25 ) by extensive dialysis against nmr sample   buffer ( 40 mm d4-malonic acid [ cambridge isotope labs , andover , ma ] ,   ph 6.0 , 0.2 mm edta , 0.2 mm dtt , and 3 mm nan3 ) .
protein concentration of the polymerized cage complex was determined using the bradford reagent ( bio - rad laboratories , hercules , ca ) . for preparation of cages composed of clathrin or clathrin and ap2 ,
an additional anion exchange chromatography step was performed to separate clathrin from ap2 before gel filtration .
ap-2 containing fractions from the second anion exchange step were then pooled   on the basis of coomassie blue  stained sds - page .
the clathrin fraction   was divided in half , and all of the ap-2 sample was added to one half of   the clathrin .
equal total protein amounts of   clathrin or clathrin / ap-2 cages were added to each nmr sample , which   contained clathrin ( 9.7 mg / ml ) or clathrin ( 8.5 mg / ml ) and ap-2 ( 1.2 mg/ ml ) .
sample purity was confirmed by coomassie blue  stained sds - page   gels and by western blotting with an antibody against -adaptin ( 100/1 ;   sigma chemical co. , st .
the clathrin terminal domain ( residues 1579 ) was expressed as a glutathione - s - transferase ( gst ) fusion protein in escherichia coli bl21 cells ,   and was prepared as described ( 17 ) .
glutathione agarose beads ( sigma   chemical co. ) with the bound gst construct were then washed with   thrombin cleavage buffer ( 50 mm tris , 2.5 mm cacl2 , 150 mm nacl , ph   7.4 ) .
the beads were incubated overnight with gentle agitation in the presence of thrombin ( sigma chemcial co. ) .
dtt was added to 10 mm and   allowed to incubate an additional 1 h before eluting the cleaved terminal   domain construct from the beads .
samples were concentrated and dialyzed overnight against nmr sample buffer with 100 mm kcl . the clathrin hub construct ( residues 10741483 )
was expressed as a   hexahistidine construct in e. coli bl21(de ) cells as described ( 29 ) .
bacterial lysates were incubated with ni - nta agarose ( qiagen inc . , valencia , ca ) overnight and eluted with 250 mm imidazole in 50 mm tris , ph   7.8 .
the eluted polypeptides were then loaded directly onto an anion exchange column equilibrated in 30 mm tris , 1 mm edta , ph 7.8 , and   eluted with a linear gradient of 0.5 m kcl .
fractions were pooled on the   basis of coomassie blue  stained sds - page gels .
because the hub   polypeptide aggregated in the nmr sample buffer , both the terminal domain and hub polypeptides were dialyzed against 40 mm kpo4 , 150 mm   kcl , 0.2 mm edta , 3 mm nan3 , and 0.2 mm dtt ph 7.2 .
all spectra were acquired at 500 mhz and 25c in sample buffer containing 10% d2o on a varian unity 500 spectrometer with a 5999.7-hz spectral width using 3-(tetramethylsilyl ) propionic acid as a chemical shift reference .
1d spectra were acquired with 128 transients of 64,000 points zero   filled to 131,072 points ( see fig .
1 ) or 64 transients of 32,000 points zero   filled to 65,536 points ( see figs . 4 and 5 ) . line - broadening was measured   as the difference of peak widths at half height using the felix 2.30 ( biosym   technologies ) program .
the broadening is reported as the line - width of   the resonance peak in the presence of cages minus the line - width in the   absence of cages .
crosspeak intensities were calculated in arbitrary units   of volume using the felix 2.30 program from spectra acquired at 60- , 100- ,   140- , and 220-ms mixing times .
the spectra were acquired in phase - sensitive mode ( 48 ) with 2  256 fids of 1024 complex points each . a spectral   width of 5999.7 hz
the relative kd for wild - type and position 807 mutant peptides from   clathrin terminal domains was obtained from measurements of the peak   intensities of the phe ( 3 , 5 ) aromatic protons for each of the peptides in   the presence and absence of protein .
the free and bound populations   were calculated from the following equation : \documentclass[10pt]{article } 
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 \begin{document } 
 \begin{equation*}\frac{1}{i_{o}}=\frac{p^{f}}{i_{f}}+\frac{p^{b}}{i_{b}}\end{equation*}\end{document }   where i is the intensity of the free ( if ) , the bound ( ib ) , and observed ( io )   resonance , and p and p are the free and bound populations , respectively .   since peak intensities
are inversely proportional to the transverse relaxation rate ( 1/t2 ) , the population of the free and bound peptide can be estimated from the change in intensity of a given peptide resonance upon addition of clathrin terminal domain if the t2 of the resonance in the free   and bound forms is known .
the t2 for a protein is approximately proportional to the molecular weight , and from the line - widths observed in 20-kd   proteins ( 3 ) , the expected t2 for a 55-kd protein is 10 ms .
the t2 calculated from the line - widths of several resonances of the free peptides was   100 ms .
the values of ib were estimated to be if/10 , assuming that the t2s   of the free and bound resonances are 100 and 10 ms , respectively .
therefore , the calculated populations of p and p were then used to obtain kds   assuming standard protein  ligand equilibrium . since the kds were based   on t2 estimates , their absolute values are likely to be inaccurate .
however , the relative values of the kds can be compared because they were all   obtained by the same method .
distances were calculated only for interresidue noes seen in 60 ms   mixing time data sets .
sequential h to nh noes were not used because   they contained contributions from the free peptide .
noe distance restraints were calculated from both wild - type hexa- and decapeptide either   by using bins of weak ( w ; 1.82.3  ) , medium ( m ; 1.83.5  ) , and strong   ( s ; 1.85.0  ) peaks , or by measured noe volumes calibrated individually   for each dimension with the average volume of the phe , tyr , and pro   ( / ) noes corresponding to 1.75  .
structures were calculated by simulated annealing performed with the programs insightii and nmrchitect ( biosym technologies , san diego , ca ) incorporating either type   of restraint . in short
, peptides were heated to 1,000 k , and then the distance and chiral restraints were slowly increased over 30 ps .
the peptides   were then slowly cooled to 300 k while increasing covalent and nonbond   forces over 32 ps .
the final structures were then obtained by iteratively   minimizing to a final derivative of 0.01 kcal / mol /  and simulating 30   ps of restrained dynamics .
removal of pseudoatom   restraints for tyr (  ,  ) and phe( ) was based on the geometry of an initial   set of structures . the final set of 30 restraints was ( * denotes pseudoatom   and distance is denoted as s , m , or w ) : ac(me*)::f(h1 ) , w ; f(h1 ) : : d(hn ) , w ; f(h2)::d(hn ) , w ; d(h2)::n(hn ) , w ; n(h1)::p(hs ) , w ;   n(h2)::p(hs ) , m ; n(h1)::p(hr ) , w ; n(h2)::p(hr ) , w ; n(hn ) : : p(hr ) , w ; p(hr)::v(hn ) , m ; p(hs)::v(hn ) , w ; p(hs)::v(hn ) , w ;   v(hn)::y(hn ) , s ; v(hn)::y(h1 ) , w ; v(h1*)::y(h1 ) , w ; v(h1 * ) : : y(h1 ) , w ; ac(me*)::d(hn ) , w ; ac(me*)::n(h21 ) , w ; ac(me * ) : : n(h22 ) , w ; v(hn)::n(h1 ) , w ; v(hn)::n(h2 ) , w ; n(h1)::y(h2 ) , w ;   n(h2)::y(h1 ) , w ; p(hs)::y(h1 ) , w ; p(hs)::y(h1 ) , w ; p(hs ) : : y(hn ) , m ; p(hr)::y(hn ) , w ; p(hr)::y(h1 ) , w ; p(hr)::y(h1 ) , w ;   p(h)::y(hn ) , w.
clathrin triskelions and aps purified from bovine brain   were assembled into membrane - free polyhedral cages using standard methods ( 25 ) .
we prepared synthetic hexa-   and decapeptides corresponding to the wild - type ldl   receptor internalization motif ( fdnpvy and fdnpvyqktt ) and to an internalization defective motif ( fdnpvs and fdnpvsqktt ) .
1 , g and h ) side chain regions from one- dimensional h nmr spectra are shown for peptide samples ( 2.5 mm ) mixed with nmr sample buffer alone , or   buffer containing clathrin cages ( 20 mg / ml ) . the wild - type   and mutant peptides exhibited characteristic narrow h   line - widths in the absence of cages ( fig .
1 , a , d , and g ,   and data not shown ) . adding cages to wild - type hexa- and  
decapeptides caused a pronounced increase in the line   widths , indicative of an interaction between the peptide   and the cages and a consequent slower effective tumbling   time for the peptide ( fig . 1 , b , c , and h ; 36 ) .
by contrast ,   the cage - induced line - broadening of mutant peptides was   markedly less than that of wild - type for both the hexa- and   decapeptides , indicating a weaker interaction ( fig . 1 , e   and f ) . as a control for nonspecific binding or viscosity - induced broadening , we examined the decapeptides in the   presence of 20 mg / ml bsa . neither peptide displayed appreciable line - broadening ( data not shown ) .
noe spectroscopy ( noesy ) is another method of detecting interactions between peptides and large protein   complexes ( 26 ) .
the hexa- and decapeptides have inefficient cross - relaxation due to their small size , and thus have   noe spectra relatively devoid of cross - peaks ( data not   shown ) .
a peptide interacting with a large protein complex will develop stronger noes because cross - relaxation   is more efficient in the bound state .
the resulting spin   populations are then maintained in the free state where   they are observed as an increase in the number and intensity of noe crosspeaks ( 10 , 11 ) . as shown in fig .
2 ( a and   c ) , the noesy spectra of both the deca- and the hexa-   wild - type peptides in the presence of cages contain numerous cross - peaks , many of which are strong . in contrast ,   only a few cross - peaks are observed in the noesy spectra of the mutant peptides in the presence of cages ( fig .
the large difference in number and intensity of   noes at a short mixing time ( 60 ms ) is consistent with the   line - broadening results ( fig .
1 ) , and indicates that the   wild - type peptides have higher affinity for the cages .  
transferred noes were not observed with either wild - type or mutant decapeptide in solutions with 20 mg / ml   bsa , even at long mixing times ( 300 ms ; data not shown ) .  
when a peptide binds to a macromolecule , the effective   tumbling rates are slower for protons in the region of the   peptide involved in binding , since protons in other regions   are likely to experience fast internal motions .
thus , cross - relaxation is more efficient in the regions immobilized by   binding , resulting in faster noe buildup rates for a given   fixed distance . to analyze the mobility of residues of the   wild - type decapeptide bound to the cages , we first compared the build - up rates of transferred noes corresponding to covalently fixed equivalent proton distances .
the   similarity in noe buildup rates ( measured with 60- , 100- ,   140- , and 220-ms mixing times ) for the geminal  protons   from residues f , n , p , and y indicates that these residues   behave isotropically in the bound state ( fig .
this   phenomenon was also apparent in the similar line - broadening of the tyr ( h , 3.45 hz ) and phe ( h , 3.60 hz ) ring   protons ( fig .
1 ) . by contrast , the buildup rates for h/h   noes decrease towards the cooh terminus of the peptide .
val has the most rapid buildup rate , indicating that it   is immobilized the most , then thr9 , and then thr10 ( fig .
this trend was also observed in line - broadening of the   val ( h1 , 5.20 hz ; h2 , 3.82 hz ) , thr9 ( h , 3.18 ) , and   thr10 ( h , 2.95 hz )  protons ( compare fig .
1 , g and h ) .   these results indicate that the decapeptide residues outside the internalization motif have greater mobility than   those within the motif .
therefore , the same residues   known to be critical for internalization in vivo were immobilized in the presence of cages
.   the internalization motif peptide could be interacting   with multiple proteins in the lattice . to distinguish between the contributions of ap2 and clathrin , we prepared   clathrin cages composed of either clathrin alone or clathrin plus purified ap2 .
4 shows that the wild - type hexapeptide in the presence of clathrin cages displayed distinct line - broadening   ( compare fig .
the mutant peptide , by   contrast , showed much less line - broadening ( compare fig .   4 d with fig .
some additional line - broadening was   seen when the wild - type peptide was mixed with cages   composed of both clathrin and ap2 ( compare fig .
however , it is likely that the broadening   arose primarily from clathrin ( see discussion ) .
the mutant   peptide ( y807s ) , by contrast , showed much less line - broadening ( compare fig .
these results suggest that the ldl receptor interacts with clathrin through the fxnpxy motif during   receptor clustering in coated pits .
we examined further the interaction between the hexapeptides and clathrin using recombinant portions of the   clathrin molecule .
the terminal domain of clathrin ( residues 1579 ) expressed as a gst fusion protein was purified , and thrombin was cleaved from gst .
clathrin hubs   ( residues 10741483 ) were expressed as a hexahis - tagged   polypeptide , and were purified by a ni resin and anion exchange .
line - broadening measurements were first made   using the terminal domain at the acidic ph used in the   cage assays .
b ) seen when peptide is incubated in the presence of   clathrin cages . the mutant peptide , by contrast , displayed   much less broadening ( compare fig .
equimolar amounts of either bsa or gst under the same   conditions had no effect ( data not shown ) .
we saw a similar degree of line - broadening at neutral ph ( compare fig .  
5 f ) , but no appreciable broadening was detected when the hub region was substituted for the terminal domain at neutral ph ( compare fig .
these results show that the ldl receptor tail selectively   interacts with the terminal domain of clathrin .
the amino acid requirements at position 807 for ldl   receptor internalization ( 14 ) have been found to correlate   with the propensity of peptides from that region to form a   reverse turn ( 2 ) .
we examined position 807 mutants for   their ability to interact with clathrin terminal domains by   measuring the change in peak intensity of 1d proton resonances for each peptide . because peak heights are inversely proportional to the effective molecular weight ,   they can be used to calculate the relative binding constant   for each peptide as it interacts with the terminal domain   ( see materials and methods ) .
table i shows that the relative affinity of each peptide for the clathrin terminal domain strongly correlated with the predicted turn propensity ( 2 ) as well as the ability of receptors bearing these   mutations to internalize ldl ( 14 ) .
thus , peptides with   tyr and phe at position 807 had the highest affinity , the   greatest turn propensity , and the highest internalization   rate , while a leu at this position had an intermediate affinity and both ser and ala had the lowest affinities
.   transferred noes have been used previously to determine the conformation of peptides bound to proteins ( 26 ,   27 , 49 ) .
these results have later been confirmed from crystal structures of the complexes ( 40 , 57 , 62 ) .
we used data   from transferred noe spectra to determine the structure   of the fdnpvy motif bound to clathrin cages .
similar   noe results were obtained when cages were replaced   with recombinant terminal domains ( data not shown ) .  
structures were calculated by simulated annealing ( 37 ) using 30 nontrivial transferred noe restraints . among   these , 23 noes correspond to the residues npvy , including the strong nh / nh noe between val and tyr characteristic of a turn conformation ( fig . 6 a ) , and other short   and intermediate range , structurally diagnostic noes .
a   superposition of the peptide backbones for the 21 structures with distance violations of < 0.1  obtained among   30 calculated structures is shown in fig .
6 b. all resulting   structures had an extended nh2 terminus followed by a   type 1  turn around residues npvy .
this result is more likely to arise from a lack of proton density in the extended structure of this region than   from increased mobility , since line - broadening and transferred noe build - up rates indicate that phe is immobilized   ( see above ) .
the 21 calculated structures had a backbone   root mean square deviation of 0.61  , indicating that a single conformational form of the peptide was bound by the   cages .
based on this information , a representative structure   is shown in fig . 6 c.
the surprising conclusion of this study is that clathrin may   be the molecule in coated pits responsible for capturing   the ldl receptor during receptor - mediated endocytosis .  
four stringent criteria were used to distinguish specific   from nonspecific interactions between the clustering motif   peptide and clathrin .
first , changing tyrosine 807 to serine ,   which matches the mutation that prevents ldl receptor   clustering in coated pits ( 13 ) , substantially reduced interaction of the peptide with the cages .
second , the wild - type   peptide bound to the cages in a single conformation , and   had the predicted ( 2 , 12 ) reverse turn conformation .
third ,   binding was limited to those residues that lie within the   fxnpxy motif .
fourth , once clathrin was identified as a   candidate molecule , the wild - type peptide was found to interact with the terminal domain , but not with the hub portion of recombinant clathrin .
furthermore , the relative affinity of terminal domains for peptides with various   position 807 substitutions correlated strongly with both   the ldl receptor internalization index ( 14 ) and the predicted turn propensity ( 2 ) .
these results suggest that the   receptor internalization rates depend on the affinities of   the hexapeptide motif for clathrin , and that the affinity depends on a reverse turn conformation .
the apparent affinity we measured between the hexapeptides and clathrin terminal domains is in the same   range as that reported for the interaction between yxx   peptides and the  subunit of ap ( 4 , 44 ) .
it is hard to judge   the functional significance of these affinities . within the   cell ,
ldl receptors probably exist as multimers interacting with the rigid clathrin lattice in a two - dimensional   plane ( 52 ) . in this environment
, the receptors will have a   restricted rotational and translational mobility as they interact with a multivalent , nearly crystalline set of terminal   domains . assuming that a 76-nm  diameter clathrin - coated   vesicle contains 60 clathrin triskelions ( 21 ) , we calculated   the concentration of terminal domains to be at minimum   1.5 mm .
this result suggests the effective concentration of   terminal domains in the region where receptor cluster is   quite high , so the relatively low - affinity interactions we   have measured should be sufficient to mediate clustering .
clathrin terminal domains have been implicated before   in receptor clustering ( 17 , 18 ) .
the binding of -arrestin to   the phosphorylated cytoplasmic tail of the 2-adrenergic   receptor appears to be required for receptor migration to   coated pits .
the binding of -arrestins to clathrin is stoichiometric , clustering in coated pits is coincident with receptor internalization , and expression of -arrestin mediates internalization ( 18 ) .
the -arrestin binding site has   been localized to the first 100 amino acids of the clathrin   heavy chain ( 17 ) .
structural studies show that the terminal   domain of clathrin is optimally positioned in an assembled   lattice to interact with the cytoplasmic tails of receptors   ( 53 ) .
however , none of these studies have sufficient resolution to determine the location of the terminal domains in   an assembled clathrin coat .
although clathrin terminal domains clearly interact with   the npxy clustering motif , we have not ruled out the possibility that aps can bind to the cytoplasmic portion of the   ldl receptor , or that they can modulate the interaction of   the nxpy motif with clathrin .
the presence of ap2 in   our clathrin cage preparations moderately enhanced line - broadening of the nmr spectra compared with clathrin   alone , suggesting that the wild - type peptides may have a   higher affinity for cages containing ap2 ( fig .
the   bound peptide displayed a well - defined conformation in   the presence or absence of aps , so broadening must be   due to a single type of binding site for the hexapeptide .   therefore , most likely the enhanced broadening is not due   to introduction of additional hexapeptide binding sites   present in the aps .
our results stand in contrast to previous studies showing   that purified ap2 binds substoichiometrically to a fusion   protein containing the cytoplasmic tail of the ldl receptor ( 42 ) .
the fusion protein used in this study contained 31   residues from the cii protein , a thrombin cleavage site ,   112 residues of myosin light chain , a second thrombin   cleavage site , and the 50-residue ldl receptor tail ( 42 ) .  
interestingly , the cii fragment contains a dileucine motif   that can directly interact with aps ( 20 , 43 ) .
since a fusion   protein containing an internalization - defective ldl receptor tail ( e.g. , y807c ) was not tested , these studies did   not rule out the possibility that the interaction with ap2   was mediated by the dileucine motif .
moreover , there is   compelling in vivo evidence that ap2 does not cluster   ldl receptors .
depletion of intracellular k and hypertonic treatment of cells both cause reversible loss of clathrin lattices from the cell surface ( 22 , 28 ) , which is accompanied by ldl receptor unclustering ( 22 , 28 ) .
if receptor   clustering were primarily dependent on ap2 , then ldl   receptors should have remained clustered under these   conditions .
an important prediction from these results is that ldl   receptors should be sorted and concentrated during clathrin - coated vesicle formation regardless of whether budding occurs from golgi , endosome , or surface membranes .  
coated vesicles purified from adrenal glands are enriched   in masked ldl receptors four to fivefold ( 33 ) .
these isolated vesicles contain coated vesicle derived from both the   plasma membrane and the golgi apparatus that can be   separated on the basis of size ( 5 ) .
ligand blotting indicates   that both populations of vesicles are equally enriched in   ldl receptors relative to the plasma membrane ( 5 ) .  
therefore , even though there are no quantitative em   studies showing ldl receptors concentrated in golgi - coated vesicles , these biochemical experiments suggest   that sorting does occur
. ap2 does appear to be responsible for capturing receptors that contain yxx ( 39 ) and dileucine internalization   motifs ( 20 , 43 ) .
two - hybrid screen ( 38 , 39 ) and surface   plasmon resonance analysis ( 4 , 39 ) both show that yxx   interacts with the  chain of ap2 .
the properties of   yxx motif are distinct from those for fxnpxy since   fxnpxy motifs did not bind the  subunit , and a combinatorial library showed no preference for this motif ( 4 ) .  
dileucine motifs also bind clathrin ap2 and ap1 , yet receptors with this motif do not compete with receptors containing yxx during internalization ( 31 ) .
receptor internalization by coated pits can therefore be mediated by at   least three distinct motifs ( two tyrosine - based and one   dileucine - based ) , each of which is captured by a different   binding site in the lattice .
tyrosine phosphorylation alters the binding specificity   of both yxx and npxy , although phosphorylation is   not essential for binding to all phosphotyrosine binding   ( ptb ) domains ( 6 , 59 ) .
pyxx binds sh2 domains ( 46 )   while npxpy binds proteins that contain a ptb domain   ( 51 ) .
the structures of both pyxx/sh2 ( 16 , 54 , 55 ) and   npxpy / ptb domain ( 15 , 60 , 61 ) complexes have been   solved .
the structure of the latter confirms transferred noe data ( 61 ) showing that the tyrosine   in the bound npxpy is in a tight turn preceded by an extended region . since we found that bound fxnpxy is in a   tight turn ( fig .
6 c ) , it is possible the simple addition of   phosphate or specific expression of a ptb domain blocks   coated pit clustering .
the conformation of yxx bound   to the  chain of ap has not been determined , but if the   phosphate is also what changes binding specificity , then   the bound conformation of yxx should be an extended ,   two - pronged plug rather than a tight turn .
this study demonstrates the power of nmr spectroscopy to identify the molecules in coat protein complexes   responsible for sorting membrane proteins during vesicular traffic . at the same time
, this method provides dynamic structural information about the targeting sequence   when it is bound .
internalization motifs   can function at multiple steps along an internalization   pathway , differentially using clathrin and ap subunits to   generate different migratory patterns .
the specificity of   clathrin for fxnpxy and aps for yxx appears to be as   distinct as ptb and sh2 domains are for their phosphorylated counterparts .
yxx and dileucine motifs may target   receptors to plasma membrane , late endosome , lysosome ,   and tgn structures via combinatorial interactions with   ap complexes and possibly other membrane coats . by   contrast , the fxnpxy motif may be needed for efficient   clathrin - mediated recycling of ldl receptors between the   plasma membrane and endosomes .
based only on the primary sequence of these motifs , it has not been possible to   predict the cellular distribution of receptors .
nmr spectroscopy should be useful to characterize further the conformational determinants of sorting motifs that ultimately   determine receptor sorting patterns .
differential line - broadening of internalization peptides   in the presence and absence of clathrin - ap cages .
corresponding   regions of 1-dimensional h nmr spectra are shown at absolute   intensity for 2.5 mm peptide with or without 20 mg / ml assembled   clathrin / ap cages .
( a  f ) aromatic region of the spectrum from   wild - type peptides fdnpvyqktt alone ( a ) , fdnpvyqktt   plus cages ( b ) , fdnpvy plus cages ( c ) , and y807s mutant peptides fdnpvsqktt alone ( d ) , fdnpvsqktt plus cages ( e ) ,   and fdnpvs plus cages ( f ) .
( g and h ) upfield region of wild - type peptide , fdnpvyqktt , alone ( g ) and with cages ( h ) .  
the resonances of the aromatic protons are at 7.29 , 7.33 , and 7.38   ppm for phe and 6.81 and 7.12 ppm for tyr and for the methyl   protons at 0.83 and 0.89 ppm for val( ) , 1.25 ppm for thr9( ) ,   and 1.18 ppm for thr10( ) .
differential broadening of the wild - type and mutant   hexapeptides in the presence of assembled clathrin cages reconstituted with and without ap-2 .
corresponding regions of   1-dimensional h nmr from the aromatic region of the spectra   are shown at absolute intensity .
the indicated peptide ( final concentration 1.5 mm ) was added to buffer alone or buffer plus 9.7   mg / ml protein : fdnpvy alone ( a ) , plus clathrin cages ( b ) , plus   clathrin and ap-2 cages ( c ) , fdnpvs alone ( d ) , plus clathrin   cages ( e ) , or plus clathrin and ap-2 cages .
nmr samples were   prepared as described for fig . 1 . because of differing concentrations of peptide and protein ,
1 . differential line - broadening of internalization peptides   in the presence and absence of recombinant clathrin terminal domains and hubs .
corresponding regions of 1-dimensional h   nmr from the aromatic region of the spectra are shown at absolute intensity .
samples contained 1.0 mm peptide with or without   175 m clathrin terminal domain td(1579 ) at ph 6.2 as follows :   fdnpvy alone ( a ) , plus td(1579 ) ( b ) or fdnpvs alone ( c ) ,   plus td(1579 ) ( d ) .
the interactions of the wild - type hexapeptide ( 1 mm ) with 200 m of either td(1579 ) or clathrin   hub(10741483 ) were compared at ph 7.2 in 150 mm kcl .
samples contained : fdnpvy alone ( e ) , plus td(1579 ) ( f ) , or plus   hub(10741483 ) ( g ) .
noesy spectra of wild - type and mutant internalization motif peptides in the presence of clathrin - ap cages .
corresponding expansions from the aliphatic / amide + aromatic region of noesy   spectra are presented as contour plots at the same level from data   acquired with 60 ms of mixing time .
all samples contained 20 mg / ml   cages and 2.5 mm peptide : fdnpvy ( a ) , fdnpvs ( b ) , fdnpvyqktt ( c ) , and fdnpvsqktt ( d ) .
noe buildup curves from the sample containing the wild - type   decapeptide with cages are shown for the intraresidue h/h   noes of phe ( squares ) , asn ( diamonds ) , pro ( circles ) , and tyr   ( triangles ; a ) , and for the intraresidue h/h noes of val(h1 )   ( squares ) , val(h2 ) ( diamonds ) , thr9 ( circles ) , and thr10 ( triangles ; b ) .
crosspeak intensities were calculated in arbitrary units   of volume using the program felix 2.30 from spectra acquired at   60 , 100 , 140 , and 220 ms mixing times . 
the relative kd calculated from phe(3,5 ) aromatic proton peak intensities as described in the experimental procedures .  
using noe magnitudes as an indicator for turn propensity , scaled to that for the   npvy peptide ( 2 ) .
( a ) contour plot of the transferred   noe between tyr(hn )   ( 7.71 ppm ) and val(hn )   ( 8.07 ppm ) from the   hexapeptide with a 60-ms   mixing time . ( b ) the superimposed backbones of 21 calculated structures obtained   by simulated annealing .
( c )   a single representative structure of the acfdnpvy peptide bound to clathrin - ap   cages .
 previously the hexapeptide motif   fxnpxy807 in the cytoplasmic tail of the ldl receptor   was shown to be essential for clustering in clathrin - coated pits . 
 we used nuclear magnetic resonance line - broadening and transferred nuclear overhauser effect   measurements to identify the molecule in the clathrin   lattice that interacts with this hexapeptide , and determined the structure of the bound motif . the wild - type   peptide bound in a single conformation with a reverse   turn at residues npvy . 
 tyr807ser , a peptide that harbors a mutation that disrupts receptor clustering , displayed markedly reduced interactions . 
 clustering motif   peptides interacted with clathrin cages assembled in the   presence or absence of ap2 , with recombinant clathrin   terminal domains , but not with clathrin hubs . 
 the identification of terminal domains as the primary site of interaction for fxnpxy807 suggests that adaptor molecules are not required for receptor - mediated   endocytosis of ldl , and that at least two different tyrosine - based internalization motifs exist for clustering   receptors in coated pits . 
Materials and Methods
Peptide Synthesis
Purification and Assembly of Clathrin/AP Cages
Expression and Purification of Recombinant Proteins
NMR Spectroscopy
Results
Discussion
Figures and Tables