protein expression and purification  the cdnas encoding 
  murine talin residues 1974 - 2293 , 1974 - 2140 , and 2137 - 2293 were synthesized by 
  pcr using a mouse talin1 cdna as template and cloned into expression vector 
  pet-151/d - topo ( invitrogen ) . constructs were expressed in 
  escherichia coli bl21 star ( de3 ) , cultured either in lb or , for 
  preparation of n - labeled samples for nmr , in minimal media 
  containing 1 g of n - ammonium chloride per liter . recombinant 
  his - tagged talin 1974 - 2293 was expressed in e. coli b834 strain for 
  selenomethionine ( semet ) incorporation , and cultured in appropriate minimal 
  media . recombinant his - tagged talin polypeptides were purified by 
  nickel - affinity chromatography following standard procedures . the his tag was 
  removed by cleavage with actev protease ( invitrogen ) , and the protein was 
  further purified by anion - exchange chromatography . recombinant his - tagged 
  chicken vinculin domain 1 ( residues 1 - 258 ) was expressed using a pet-15b 
  expression plasmid and purified as described previously 
  ( 28 ) . the concentration of 
  purified proteins was determined using the cb - protein assay ( calbiochem ) . x - ray crystallography  crystals of talin residues 1974 - 2293 
  were obtained at 19 c by vapor diffusion equilibration against 10% ( w / v ) 
  polyethylene glycol 8000 , 100 mm hepes , 1% ( w / v ) polyethylene 
  glycol 3350 , 10 mm sodium thiocyanate at ph 7.5 . protein at 5.0 
  mg / ml in 0.2 m nacl , 2 mm dithiothreitol , and 20 
  mm tris - hcl , ph 8.0 , was mixed with an equal volume of precipitant . native protein yielded form 1 crystals , with 1 molecule per 
  asymmetric unit , whereas semet crystals yielded form 2 crystals containing 2 
  molecules per asymmetric unit ( see table 
  1 ) , with solvent contents of 47% and 45% , respectively . the two 
  forms are closely related in their crystal packing , but there is a near 
  doubling of the c - axis in form 2 to accommodate the second 
  molecule . table 1summary of crystallographic analysis and refinement statistics for talin 
  residues 1974 - 2293rsym = s|i - < i>|si , where i is the 
  observed intensity and < i > is the average intensity of the multiple 
  observations of symmetry - related reflections . r = 
  s||fo| - 
  |fc||/s|fo| ; 
  rfree is calculated for a randomly selected 5% number of 
  the reflections ; rfactor is calculated for the remaining 
  95% of the reflections used in refinement . values in parentheses represent the 
  outer resolution shell.data collection space group 
   p21 p21 cell dimensions 
  a = 59.4 
  a = 58.7  
  b = 57.1  
  b = 57.6  
  c = 92.1  
  c = 92.6  
    = 102.8 
    = 103.0 
   no . of molecules in asymmetric unit 
   2 
   2 
   data set 
   peak 
   high resolution 
   wavelength (  ) 
   0.9791 
   0.9757 
   resolution (  ) 
   20 - 2.5 
   20 - 1.85 
   measured reflections 
   154011 
   559438 
   unique reflections 
   40327 
   52439 
   completeness ( % ) 
   99.8 ( 99.5 ) 
   98.1 ( 87.5 ) 
  rsym 9.0 ( 30.6 ) 
   8.5 ( 44.9 ) 
 
  i/i 
 
  12.8 ( 5.7 ) 
 
  16.8 ( 1.7 ) 
  refinement statistics resolution range (  ) 
   20 - 1.85 
   unique reflections ( free ) 
   49457 
  rwork ( % ) 
   21.4 ( 29.0 ) 
  rfree ( % ) 
   26.0 ( 41.0 ) 
   number of residues / atoms 
   632/5010 
   number of solvent molecules 
   332 
   average b value (  ) 
   31 
   r.m.s.d . bond angles (  ) 
 
  1.45 
   summary of crystallographic analysis and refinement statistics for talin 
  residues 1974 - 2293 rsym = s|i - < i>|si , where i is the 
  observed intensity and < i > is the average intensity of the multiple 
  observations of symmetry - related reflections . r = 
  s||fo| - 
  |fc||/s|fo| ; 
  rfree is calculated for a randomly selected 5% number of 
  the reflections ; rfactor is calculated for the remaining 
  95% of the reflections used in refinement . diffraction data were collected from native crystals ( form 1 ) at european 
  synchrotron radiation facility beamline id23 - 1 , and from semet crystal ( form 
  2 ) at beamline 14 - 4 , recorded on adsc q315r charge - coupled device detectors . phases were determined 
  from the anomalous data collected at the selenium absorption peak (  = 
  0.9791 ) from semet form 2 crystals . 10 of the 12 possible selenium atoms were 
  located ( 6 per molecule ) , and a map was constructed using these preliminary 
  phases at 2.5- resolution . an initial atomic model was built using 
  solve / resolve ( 30 ) , and , 
  following phase improvement with dm 
  ( 31 ) , the model was rebuilt 
  manually with coot ( 32 ) and 
  refined using maximum likelihood refinement in refmac5 
  ( 33 ) . subsequently , the 
  structure was refined against a new 1.85- data set collected from form 
  2 semet crystals . the final model converged to an rwork of 21.4% for all 
  data between 20 and 1.85  , and an rfree of 26.0% . the final ramachandran plot shows 96.9% of residues in favored regions , 2.8% 
  in additional favored regions , and 0.2% in generously allowed regions , as 
  defined by procheck ( 34 ) . the 
  structure has been submitted to the protein data bank with the accession 
  number 3dyj 
  ( www.rcsb.org ) . the form 1 native 
  crystals did not diffract as well as the semet derivatives , and the data were 
  partially refined to an rfree of 23.7% . gel filtration and proteolysis  analytical gel filtration 
  chromatography of recombinant talin fragments 1974 - 2293 , 1974 - 2140 , and 
  2137 - 2293 , as well as vinculin vd1-(1 - 258 ) , was performed using superdex-75 
  ( 10/300 ) gl ( amersham biosciences ) at room temperature . the proteins were 
  mixed and incubated at various temperatures for 30 min prior to loading onto 
  the column , which was pre - equilibrated with and run with 20 mm 
  tris , ph 8.0 , 150 mm nacl , and 2 mm dithiothreitol at a 
  flow rate of 0.8 ml / min . all proteolysis experiments were carried out at 20 
  c for 1 h using a 1:50 ( w / w ) trypsin : protein ratio . saxs  small angle x - ray scattering ( saxs ) experiments were 
  carried out at station 2.1 of the u.k . synchrotron radiation source at 
  daresbury , using a multiwire gas detector covering a momentum - transfer range 
  of 0.02  < q < 0.70  , 
  where q = 4 sin / ( 2 is the scattering 
  angle and  the x - ray wavelength , 1.54  ) . measurements on talin 
  1974 - 2293 were performed at 4 c at concentrations of 2 and 10 mg / ml in a 
  buffer comprising 20 mm sodium phosphate , ph 6.5 , 50 mm 
  nacl , and 2 mm dithiothreitol . experimental data were accumulated 
  in 60-s frames , and , before averaging , frames were inspected for x - ray - induced 
  damage or aggregation . the background was subtracted using the scattering from 
  the buffer solution alone . no protein aggregation was detected , and the 
  linearity of the guinier plot ( supplemental fig . data reduction was carried out with 
  software provided at the daresbury facility , and subsequent analysis was done 
  with the atsas program package 
  ( 36 ) . the theoretical 
  rg for the crystal structure of the ibs2 domain was 
  calculated using crysol 
  ( 37 ) . nmr spectra were collected using a 0.2 
  mm protein solution in 20 mm sodium phosphate buffer ( ph 
  6.5 ) , 50 mm nacl , and 2 mm dithiothreitol at 298 k on a 
  bruker avance drx600 spectrometer equipped with a cryoprobe . binding of integrin tails to talin rod using a pulldown 
  assay  purified talin rod fragments were diluted to 250 
  nm in pn buffer ( 10 mm pipes , 50 mm nacl , 150 
  mm sucrose , 50 mm naf , 40 mm sodium 
  phosphate , ph 6.8 ) . to assay for integrin binding , 700 l of protein 
  solution was mixed ( incubated for 2 h at room temperature ) with 10 g of 
  his - avi - tagged integrin tails immobilized on neutravidin - coated beads . unbound 
  proteins were removed by three washes in pn buffer containing 5% triton x-100 , 
  and bound proteins were solubilized in laemmli sample buffer and detected by 
  western blotting ( 38 ) . mouse 
  anti - v5 antibody ( invitrogen ) was used to detect talin rod domains , and a 
  rabbit anti - his antibody ( santa cruz biotechnology ) was used to detect the 
  talin head . microtiter 
  wells ( elisa high binding plate , white , fisher ) were coated with a solution of 
  10 g / ml neutravidin ( pierce ) and incubated with 150 l of blocking 
  buffer ( 1% heat inactivated - bovine serum albumin in pbs ) . wells were then 
  incubated with purified recombinant his - avi - tagged integrin tails ( 2 g / ml ) 
  diluted in pbs containing 1% bovine serum albumin and 0.2% tween 20 ( sample 
  buffer ) . talin rod fragments ( 25 - 2500 nm ) in sample buffer were 
  added to the wells , and bound talin was detected with primary mouse anti - v5 
  antibody ( invitrogen , ratio 1:5000 ) and a secondary goat anti - mouse 
  horseradish peroxidase - conjugated antibody ( biosource ) using luminescence with 
  ecl ( amersham biosciences ) . all incubations were for 1 h at 37 c in 50 
  l of buffer unless otherwise stated , and plates were washed three times 
  with pbs containing 0.2% tween 20 after each step . controls included wells 
  without neutravidin , neutravidin without integrin , and wells coated with 
  iib - integrin . integrin loading onto neutravidin plates was quantitated 
  using mouse 7h8 anti - helix monoclonal antibody , and some wells were coated 
  with the talin fragments to verify equal loading of the various 
  constructs . vinculin - null mouse embryonic fibroblasts 
  ( 39 ) cultured in dulbecco 's 
  modified eagle 's medium containing 10% fetal calf serum with 2 mm 
  l - glutamine were plated onto glass coverslips , and 24 h later 
  transfected with cdnas encoding various pegfp - c2-tagged mouse ibs2 
  polypeptides using fugene 6 ( roche applied science ) , according to the 
  manufacturer 's instructions . cells were fixed with pre - warmed 
  para - formaldehyde ( 4% ( w / v ) in pbs ) , permeabilized with 0.5% triton 
  x-100 in pbs , and stained using a mouse monoclonal antibody to paxillin ( clone 
  349 , bd biosciences , diluted 1:300 in 0.05% triton x-100 in pbs ) followed by a 
  goat anti - mouse conjugated to alexafluor 568 fluorescent dye . cells were 
  imaged using an inverted zeiss axiovert 200 m microscope equipped with a 
  63 oil immersion lens ( numerical aperture = 1.3 ) . phospholipid binding  phosphatidylinositol phosphate strips 
  ( invitrogen ) were treated at room temperature for 5 h with 3% ovalbumin in 
  tbs - t ( 10 mm tris , ph 8.0 , 150 mm nacl , 0.1% tween 20 ) 
  to eliminate nonspecific binding , and incubated overnight at 4 c with 1 
  g / ml talin fragments in tbs - t containing 3% ovalbumin . after incubation , 
  the strips were washed three times at room temperature in tbs - t containing 
  0.1% ovalbumin , and talin binding was detected with a mouse anti - his 
  horseradish peroxidase - conjugated antibody ( ratio 1:6000 , 1 h at room 
  temperature , alpha diagnostics ) , followed by three washes in tbs - t . peptides ( 25- and 36-mers ) based on the mouse 
  -integrin sequences were synthesized on a fully automated spot 
  synthesizer multipep ( intavis ag , germany ) . the derivatization of hydroxyl 
  groups of cellulose sheets ( schleicher & schuell , germany ) was carried out 
  with fmoc - alanine , 1-methylimidazole , and dicyclohexylcarbodiimide in dimethyl 
  formamide overnight . the peptides were then synthesized by repeated deposition 
  of pre - activated amino acids onto derivatized cellulose sheets via fmoc 
  chemistry , using dicyclohexylcarbodiimide/1-hydroxybenzotriazole activation of 
  amino acids in n - methyl-2-pyrrolidone and fmoc deprotection with 20% 
  ( v ) piperidine in dimethyl formamide . during the first three cycles of 
  synthesis , residual amino groups and the final n - terminal amino groups were 
  blocked with a mixture of 80% acetic anhydride/10% 
  n , n - diisopropylethylamine/10% dimethyl formamide ( v / v ) for 30 min . finally , cleavage of side - chain protection groups was carried out in 95% 
  trifluoroacetic acid/5% dichloromethane ( v / v ) for 45 min . talin ibs2 binding to -integrin spot - peptide 
  arrays  membranes were treated overnight with 10% fetal bovine serum 
  in tris - buffered saline ( 50 mm tris - hcl , ph 7.0 , 137 mm 
  nacl , 2.7 mm kcl ) . murine talin fragment c ( residues 1975 - 2541 ) , 
  n - terminally tagged with t7 & gfp , and c - terminally with his-7 , was 
  expressed using pet23a - t7 ( 40 ) 
  and purified on nickel - nitrilotriacetic acid - sepharose ( qiagen ) according to 
  the manufacturer 's protocol . eluted protein was subjected to mono q 
  ion - exchange chromatography ( amersham biosciences ) , and purified t7/gfp - talin 
  c was transferred into pbs . membranes were overlaid for 2 h with t7/gfp - talinc 
  ( 1 m ) in tris - buffered saline with 1% bovine serum albumin at 
  room temperature . bound t7/gfp - talin fragment c was detected using a 
  monoclonal t7 antibody ( novagen ) and alkaline phosphatase - coupled anti - mouse 
  ig ( jackson laboratories ) , as described previously 
  ( 3 ) . differential scanning calorimetry  dsc experiments were 
  carried out using an ndsc ii calorimeter ( csc ) at a scanning rate of 1 k / min 
  under 3.0 atm of pressure . protein samples were exchanged into dsc buffer 
  comprising 20 mm pipes ( ph 7.5 ) and 100 mm nacl . sven 
  rothemund ( interdisziplinres zentrum fr klinische forschung 
  leipzig ) , and also dissolved in dsc buffer . protein samples were analyzed at 
  0.7 mg / ml with -integrin peptide at 100 or 200 m . crystal structure of talin ibs2 ( residues 1974 - 2293)to 
  determine the structure of ibs2 , we expressed recombinant talin residues 
  1974 - 2293 in e. coli , and obtained crystals from the purified 
  polypeptide that diffracted x - rays . we determined initial phases using 
  anomalous scattering from a selenomethionine derivative from form 2 crystals 
  ( see  experimental procedures  ) at 2.5- resolution , and 
  subsequently collected a diffraction set to 1.85- resolution for high 
  resolution refinement ( table 
  1 ) . the final high resolution model includes two copies of ibs2 
  ( residues 1975 - 2291 ) within the asymmetric unit . ibs2 comprises a tandem pair 
  of five - helix bundles with the same topology , comprising five anti - parallel 
  -helices ( ranging from 24 to 30 residues in length ) . helices 
  2-5 and 7-10 are folded into left - handed 
  up - down - up - down 4-helix bundles . in both cases , a long 10-residue linker 
  connects the first and second helices ( i.e. 1-2 and 
  6-7 ) ; otherwise , the helices are connected by short loops 
  ( fig . 2a and 
  supplemental fig . the two ibs2 five - helix bundles can be superposed using 
  coot ( 32 ) with an r.m.s.d . of 
  2.4  on backbone atoms ( supplemental fig . this bundle 
  topology has only previously been seen ( dali 
  ( 41 ) ) in the five - helix bundle 
  at the n terminus of the talin rod ( residues 482 - 655 ) 
  ( 28 ) , and both ibs2 bundles 
  superpose with this domain with an r.m.s.d . figure 2.structure of ibs2 in the talin rod . a , schematic 
  representation of the talin 1974 - 2293 crystal structure . the upper five - helix 
  bundle is called ibs2-a , and the lower one ibs2-b . b , stereo representation 
  of the area located between the two domains in the crystal structure ; there is 
  no evidence of hydrophobic or electrostatic interactions between the two 
  domains . the upper five - helix 
  bundle is called ibs2-a , and the lower one ibs2-b . b , stereo representation 
  of the area located between the two domains in the crystal structure ; there is 
  no evidence of hydrophobic or electrostatic interactions between the two 
  domains . the two bundles are linked by an almost continuous helix , but with a 
  distinct kink between the bundles . several intrahelical main - chain h - bonds are 
  lost between residues glu-2138 and thr-2143 , dividing the helix into two 
  segments ( 5 and 6 ) , which are assigned to the n- and c - terminal 
  bundles , respectively . the kink is , however , stabilized by several intra- and 
  interhelical h - bonds that replace the lost main - chain h - bonds 
  ( fig . three interactions with the beginning of helix 8 
  stabilize the kink : the amide side chain of gln-2198 makes simultaneous 
  h - bonds with the  orphan  amides of gly-2142 and thr-2143 , whereas 
  the side chains of thr-2140 and arg-2144 act as c - terminal caps to helix 
  5 . in addition , the side chain of glu-2199 makes a salt bridge with 
  lys-2141 , and the side chain of ile-2202 packs against gly-2142 . these 
  features are conserved in all known sequences and are structurally conserved 
  in all three copies of the two crystal forms , suggesting that the helical kink 
  is a biological feature of the two - domain module . other contacts between the 
  bundles are limited and vary in different crystal environments ( see 
  below ) . the two molecules ( a and b ) in the form 2 asymmetric unit have similar 
  tertiary and quaternary organizations , but there are some significant 
  differences . thus , the individual bundles overlay with r.m.s.d . values of 
  < 0.5  for most main - chain residues . however , the large 
  1-2 loop in the first bundle adopts two distinct conformations , 
  beginning at residue asn-2005 and propagating down helix 2 as far as 
  lys-2024 . at the apex of the 1-2 turn , residue ala-2009 in 
  molecule a shifts by 5.5  toward the second bundle compared with 
  molecule b so that it makes hydrophobic contacts with the 9-10 
  loop from the second bundle ; there are also several water - mediated polar 
  interaction ; nevertheless , the interface is limited . in molecule b , the only 
  significant contacts between the two domains are two long range hydrogen bonds 
  ( arg-2006 h - bonds to the c = o of gln-2259 , and lys-2260 h - bonds to gly-2008 
  c = o ) . the interfacial difference is linked to a significant alteration in the 
  quaternary organization of the module , involving a 2- to 3- translation 
  of the second bundle with respect to the first in the two molecules . this is 
  accommodated by a gradual bend in helix 5 with little change in the 
  kink angle ( 40 ) . in form 1 crystals ( see  experimental 
  procedures  ) , the intrabundle contacts closely resemble those of form 2 
  molecule a. furthermore , analysis of crystal contacts shows that this region 
  in form 2 molecule b forms several lattice contacts , whereas molecule a does 
  not . a , talin polypeptides spanning residues 1974 - 2293 ( ibs2 ) , 1974 - 2140 
  ( ibs2-a ) , and 2137 - 2293 ( ibs2-b ) were analyzed on a superdex-75 ( 10/300 ) gl 
  gel filtration column . the apparent molecular mass for each domain is 
  indicated with their theoretical molecular mass in brackets . the 
  talin ibs2 polypeptides showed an anomalous elution profile indicative of an 
  extended conformation . b , saxs of the talin ibs2 polypeptide 
  indicates a different domain organization from that in the crystal structure . experimental scattering profile of talin ibs2 ( red ) compared with the 
  simulated scattering profile based on the crystal structure ( black 
  line ) ( goodness - of - fit = 8.9 ) . c and d , binding of 
  the vinculin vd1 domain to talin ibs2 ( c ) or ibs2-a ( d ) was 
  analyzed on a superdex-75 ( 10/300 ) gl gel filtration column at room 
  temperature ( rt ) . incubation of either ibs2 or ibs2-a with vd1 at 
  room temperature resulted in rather little complex formation , and most of the 
  talin and vinculin polypeptides remained in the free form . however , 
  preincubation of the proteins at 37 c resulted in formation of a 
  talin - vd1 complex . a , talin polypeptides spanning residues 1974 - 2293 ( ibs2 ) , 1974 - 2140 
  ( ibs2-a ) , and 2137 - 2293 ( ibs2-b ) were analyzed on a superdex-75 ( 10/300 ) gl 
  gel filtration column . the apparent molecular mass for each domain is 
  indicated with their theoretical molecular mass in brackets . the 
  talin ibs2 polypeptides showed an anomalous elution profile indicative of an 
  extended conformation . b , saxs of the talin ibs2 polypeptide 
  indicates a different domain organization from that in the crystal structure . experimental scattering profile of talin ibs2 ( red ) compared with the 
  simulated scattering profile based on the crystal structure ( black 
  line ) ( goodness - of - fit = 8.9 ) . c and d , binding of 
  the vinculin vd1 domain to talin ibs2 ( c ) or ibs2-a ( d ) was 
  analyzed on a superdex-75 ( 10/300 ) gl gel filtration column at room 
  temperature ( rt ) . incubation of either ibs2 or ibs2-a with vd1 at 
  room temperature resulted in rather little complex formation , and most of the 
  talin and vinculin polypeptides remained in the free form . however , 
  preincubation of the proteins at 37 c resulted in formation of a 
  talin - vd1 complex . solution studies support the crystallographic studies , pointing to an 
  extended conformation with some flexibility , as judged by analytical gel 
  filtration , saxs , and nmr ( fig . 3 
  ( a and b ) and supplemental fig . nmr line - widths point to a module of intermediate flexibility : the increase in 
  line width of the ibs2 module compared with the individual bundles 
  ( supplemental fig . s4 , a - c ) is greater than that expected for a pair 
  of domains tumbling independently , but it is also greater than that expected 
  for a rigid domain pair . additionally , the positions of the resolved 
  resonances change very little compared with the two domains in isolation , 
  consistent with the small interfacial area demonstrated 
  crystallographically . the ibs2 module is relatively resistant to trypsin digestion , despite its 
  high arg and lys content ( 11% ) . the most abundant cleavage site is at lys-2133 
  between the two five - helix bundles , whereas lys-2141 and arg-2144 , which are 
  also surface - exposed in the crystal structure , are resistant to cleavage . the 
  crystal structure rationalizes these data : thus lys-2133 is fully exposed and 
  makes only a weak ionic interaction with asp-2137 
  ( fig . 2b ) ; by 
  contrast , lys-2141 is sandwiched between two glutamates , glu-2138 and 
  glu-2139 , whereas arg-2144 forms multiple intramolecular interactions 
  contributing to the 5 helix cap , as noted above . ibs2 binds the 1 and 3 integrin 
  cytoplasmic domains  to confirm that the c - terminal region of the 
  talin rod interacts with integrin cytoplasmic tails , we first performed 
  pulldown experiments using biotinylated integrin tails immobilized on 
  neutravidin beads . ibs2 , as well as two longer constructs , 1974 - 2482 and 
  1974 - 2541 ( the latter includes the c - terminal dimerization domain ) , all bound 
  strongly to integrin 3 , whereas the c - terminal domain alone ( residues 
  2300 - 2541 ) bound weakly ( fig . 4 , a 
  and b ) . these data are consistent with the 
  integrin - binding site being located within ibs2 . in contrast to previous 
  reports , we found that individual ibs2a and ibs2b bundles bound integrin 
  weakly or not at all . ibs2 also bound weakly to the iib - integrin tail , 
  although its significance is unclear . figure 4.the talin 1974 - 2293 ibs2 polypeptide binds to 3-integrin 
  tails in pulldown and elisa - type assays . a , schematic of talin 
  rod polypeptides used where each box represents a five - helix bundle 
  with the exception of the c - terminal dimerization domain , which is composed of 
  a single -helix that forms an anti - parallel dimer 
  ( 16 ) . all constructs include 
  an n - terminal his tag followed by a v5 epitope . b , pulldown assays 
  using iib- and 3-integrin tails immobilized on neutravidin beads 
  with purified recombinant talin rod polypeptides . binding of talin rod 
  polypeptides was detected using an anti - v5 antibody , while binding of the 
  talin head ( used as a positive control ) was detected using an anti - his 
  antibody ( data not shown ) . c and d , binding of talin 
  polypeptides to microtiter wells coated with 3-integrins using elisa . 
  the talin head polypeptide ( residues 1 - 405 ) was used as a positive control . 
  binding to wells coated with iib - integrin or the talin ibs2 polypeptide , which contains 
  both the ibs2-a and ibs2-b five - helix bundles , binds to 3-integrin with 
  much higher affinity than the individual ibs2-a and ibs2-b five - helix 
  bundles . the talin 1974 - 2293 ibs2 polypeptide binds to 3-integrin 
  tails in pulldown and elisa - type assays . a , schematic of talin 
  rod polypeptides used where each box represents a five - helix bundle 
  with the exception of the c - terminal dimerization domain , which is composed of 
  a single -helix that forms an anti - parallel dimer 
  ( 16 ) . all constructs include 
  an n - terminal his tag followed by a v5 epitope . b , pulldown assays 
  using iib- and 3-integrin tails immobilized on neutravidin beads 
  with purified recombinant talin rod polypeptides . binding of talin rod 
  polypeptides was detected using an anti - v5 antibody , while binding of the 
  talin head ( used as a positive control ) was detected using an anti - his 
  antibody ( data not shown ) . c and d , binding of talin 
  polypeptides to microtiter wells coated with 3-integrins using elisa . the talin head polypeptide ( residues 1 - 405 ) was used as a positive control . 
  binding to wells coated with iib - integrin or not coated with integrins 
  were used as negative controls . the talin ibs2 polypeptide , which contains 
  both the ibs2-a and ibs2-b five - helix bundles , binds to 3-integrin with 
  much higher affinity than the individual ibs2-a and ibs2-b five - helix 
  bundles . to further characterize these interactions , we used an elisa - type assay in 
  which biotinylated integrin tails were immobilized on neutravidin - coated 
  microtiter wells . binding of v5-tagged talin rod polypeptides was quantified 
  using an anti - v5 monoclonal antibody . talin head was used as a positive 
  control , and uncoated neutravidin - treated wells as negative controls . talin head ( residues 1 - 405 ) bound in 
  a dose - dependent manner to 3-integrin tail 
  ( fig . 4c ) with an 
  ec50 of 0.4  0.2 m , consistent with 
  published surface plasmon resonance studies 
  ( 23 ) . the long construct , 
  talin 1974 - 2541 , bound with lower affinity ( ec50 > 2.5 
  m ) , consistent with published data on the talin rod 
  ( 23 ) . however , the ibs2 module 
  alone bound with significantly higher affinity ( ec50 0.9  
  0.2 m ) , comparable to that of the head , suggesting that 
  elements c - terminal to the ibs2 module may be autoinhibitory . a very similar 
  ec50 was found for binding of ibs2 to 1a - integrin ( 0.9 
   0.1 m ) . the individual bundles of ibs2 did show 
  dose - dependent integrin binding ( fig . 4d ) , but it was weak and not saturable under the 
  conditions employed , with estimated ec50 values of > 2.5 
  m . these results confirm that strong integrin binding requires 
  the intact ibs2 module . ibs2-a contains a potential vinculin - binding site in helix 
  4 ( talin rod helix 50 ) 
  ( 3 ) , but , as with all such 
  sites , the vinculin - binding residues are buried in the hydrophobic core of the 
  bundle . very little binding between the ibs2 module and vinculin d1 domain 
  ( vd1 ) was observed at room temperature , but incubation at 37 c led to 
  substantial complex formation as detected by gel filtration 
  ( fig . we have 
  previously shown a similar temperature dependence for the vinculin - binding 
  site in talin 482 - 655 ( 28 , 
  42 ) . ibs2 has a 
  tm of 58 c , and we conclude that increasing the 
  temperature to the more physiologic 37 c has a destabilizing effect on 
  bundle integrity that facilitates vinculin binding . as expected , only the 
  ibs2-a bundle bound vd1 ( fig . talin fragments from the ibs2 region have been shown to 
  localize to focal adhesions ( fas ) 
  ( 25 , 
  43 ) , but because ibs2a 
  contains a vinculin - binding site 
  ( 3 ) , it was unclear whether 
  localization reflected binding to integrin or vinculin . to test this , we 
  expressed gfp - tagged ibs2 in vinculin - null mouse embryo fibroblasts and found 
  that it co - localized efficiently to fas 
  ( fig . in contrast , gfp - ibs2-a 
  and gfp - ibs2-b displayed only a diffuse cytoplasmic fluorescence . the data 
  mirrors that on integrin binding and suggest that fa localization of ibs2 
  reflects its ability to bind integrins . because activation of integrins by the talin ferm domain is dependent on 
  interactions between basic residues on the membrane - proximal surface of the 
  ferm domain and acidic phosphoinositides in the plasma membrane 
  ( 5 ) , we explored the binding of 
  ibs2 to a range of phospholipids spotted on a nitrocellulose membrane . we 
  found that ibs2 bound strongly to certain acidic lipids , including 
  phosphatidylinositol 3,5-bisphosphate and phosphatidic acid 
  ( fig . 6 ) , and that , as in the 
  case of integrin - binding and fa targeting , the two - domain ibs2 module is 
  required . interestingly , binding to phosphatidylinositol 4,5-bisphosphate , 
  which is up - regulated at focal adhesions , was significantly weaker . mouse 
  embryonic fibroblasts derived from vinculin knockout mice 
  ( 39 ) were transfected with 
  cdnas encoding egfp - tagged ibs2 fragments . fas were visualized by paxillin 
  staining . the ibs2 double domain construct clearly localized to fas , whereas 
  the individual five - helix bundles ibs2-a and ibs2-b showed little or no 
  targeting . mouse 
  embryonic fibroblasts derived from vinculin knockout mice 
  ( 39 ) were transfected with 
  cdnas encoding egfp - tagged ibs2 fragments . the ibs2 double domain construct clearly localized to fas , whereas 
  the individual five - helix bundles ibs2-a and ibs2-b showed little or no 
  targeting . binding of his - tagged talin polypeptides to phosphatidylinositol phosphate 
  strips containing an array of acidic phospholipids ( invitrogen ) was detected 
  using an anti - his antibody . each spot contains 100 pmol of 
  phospholipid , and the membrane was challenged with 1 g / ml protein . talin 
  ibs2 ( residues 1974 - 2293 ) binds to several phospholipids , whereas the 
  individual five - helix bundles that make up ibs2 , i.e. residues 
  1974 - 2140 ( ibs2-a ) and residues 2137 - 2293 ( ibs2-b ) , did not bind to any of the 
  phospholipids tested . binding of his - tagged talin polypeptides to phosphatidylinositol phosphate 
  strips containing an array of acidic phospholipids ( invitrogen ) was detected 
  using an anti - his antibody . each spot contains 100 pmol of 
  phospholipid , and the membrane was challenged with 1 g / ml protein . talin 
  ibs2 ( residues 1974 - 2293 ) binds to several phospholipids , whereas the 
  individual five - helix bundles that make up ibs2 , i.e. residues 
  1974 - 2140 ( ibs2-a ) and residues 2137 - 2293 ( ibs2-b ) , did not bind to any of the 
  phospholipids tested . we 
  first probed filters containing three overlapping spot - synthesized 25-mer 
  peptides spanning the cytoplasmic tails of 1a , 2 , 3 , and 
  7 integrins ( fig . 7 , a and 
  b ) with an ibs2 polypeptide spanning residues 1974 - 2541 . the data locate the major ibs2-binding site within the 23 membrane - proximal 
  residues of the -integrin tails ( fig . replacement of most acidic residues led to a substantial increase in 
  ibs2 binding , whereas substitution of basic residues caused a small but 
  consistent reduction , indicating an important role for charged residues in the 
  interaction , and suggesting that the binding site on ibs2 is acidic . effects of -integrin peptides on the stability of the 
  talin ibs2 module  to further characterize integrin binding to ibs2 , 
  we used dsc to monitor the effect of integrin binding on the melting 
  temperature ( tm ) and unfolding enthalpy of ibs2 . binding 
  of the full - length 1a - integrin tail peptide ( 47-mer ) to ibs2 
  ( fig . 8a and 
  table 2 ) led to a decrease in 
  h at 100 m integrin . at 200 m 
  integrin , h was reduced further , and there was also a large 
  reduction in tm , suggestive of a conformational change and 
  perhaps partial unfolding of ibs2 . two peaks with different 
  tm values are evident in the ibs2 module , and integrin 
  binding promotes the peak with the lower melting temperature . 8 , 
  b and c ) show that the lower peak was entirely 
  attributable to ibs2-a . ibs2-b did bind integrin , as judged by a significant 
  reduction in tm and h , but still melted as 
  a single peak . binding of the shorter membrane - proximal peptide ( 25-mer ) 
  caused a similar decrease in h but did not lead to the 
  formation of the peak at lower tm 
  ( fig . 8 , d - f ) 
  suggesting that membrane - distal portions of the integrin contribute 
  significantly to binding and conformational change , especially in ibs2-a . table 2thermodynamic parameters obtained from dsc scans for talin 1974 - 2293 , 
  1974 - 2140 , and 2137 - 2293 upon binding 1a - integrin peptidesthe absolute error in tm values did not exceed 
  0.2 c ; the relative error in hcal 
  values did not exceed  10%.1974 - 2293 
 1974 - 2140 
 2137 - 2293 
 tmhtmhtmh ckcal / mol ckcal / mol ckcal / mol talin alone 
   57.9 
   119 
   58.4 
   44 
   60.3 
   76 
   talin + 100 m 1a ( 47-mer ) 
   58.0 
   94 
   58.7 
   36 
   58.7 
   63 
   talin + 200 m 1a ( 47-mer ) 
   52.4 
   67 
   talin + 100 m 1a ( 25-mer ) 
   58.1 
   90 
   58.2 
   42 
   59.1 
   59 
 
  talin + 200 m 1a ( 25-mer ) 
 
  58.2 
 
  72 
   thermodynamic parameters obtained from dsc scans for talin 1974 - 2293 , 
  1974 - 2140 , and 2137 - 2293 upon binding 1a - integrin peptides the absolute error in tm values did not exceed 
  0.2 c ; the relative error in hcal 
  values did not exceed  10% . we have determined the structure of talin residues 1974 - 2293 ( ibs2 ) , which 
  consists of two five - helix bundles connected by a long helix with a pronounced 
  kink or flexible region at its center . the topology of the two bundles is 
  identical and so far unique to talin . the modest inter - bundle interface 
  ( 600  ) comprises mostly charged residues . solution 
  studies by saxs and nmr are consistent with an extended conformation and with 
  a relatively small interface with limited influence of one bundle on the 
  other . weak interactions between domains in the talin rod are not unexpected , 
  because talin is thought to switch between a globular inactive and a more 
  extended active conformation , which must involve changes in the interactions 
  between at least some of the bundles . a relatively flexible interface and 
  end - to - end packing between the helical bundles seems to be typical of the 
  c - terminal region of the talin rod but contrasts sharply with the staggered 
  arrangement of the two bundle module at the n terminus of the rod , which is 
  stabilized by an extensive hydrophobic interface 
  ( 28 ) . figure 7.talin ibs2 binds to membrane proximal -integrin tail 
  peptides . a , alignment of the full - length 1a - integrin 
  cytoplasmic domain peptide with the membrane proximal , middle , and distal tail 
  peptides used in this study . b and c , analysis of the 
  binding of a talin 1975 - 2541 polypeptide to a series of immobilized 
  -integrin cytoplasmic domain peptides . b , binding to membrane 
  proximal ( top ) , middle , and distal tail peptides equivalent to 
  1a- , 2- , 3- , and 7-integrins ( 25-mers ) . binding to 
  membrane proximal peptides from all -integrin tails was observed 
  ( including 5 and 6 ; data not shown ) . c , analysis of 
  binding to a series of membrane proximal -integrin peptides ( 25-mers ) in 
  which each residue in turn was substituted by alanine . mutation of -integrin membrane proximal peptides identify important 
  charged residues required for optimal talin 1975 - 2541 binding ( see 
  a ) . talin ibs2 binds to membrane proximal -integrin tail 
  peptides . a , alignment of the full - length 1a - integrin 
  cytoplasmic domain peptide with the membrane proximal , middle , and distal tail 
  peptides used in this study . b and c , analysis of the 
  binding of a talin 1975 - 2541 polypeptide to a series of immobilized 
  -integrin cytoplasmic domain peptides . b , binding to membrane 
  proximal ( top ) , middle , and distal tail peptides equivalent to 
  1a- , 2- , 3- , and 7-integrins ( 25-mers ) . binding to 
  membrane proximal peptides from all -integrin tails was observed 
  ( including 5 and 6 ; data not shown ) . c , analysis of 
  binding to a series of membrane proximal -integrin peptides ( 25-mers ) in 
  which each residue in turn was substituted by alanine . mutation of -integrin membrane proximal peptides identify important 
  charged residues required for optimal talin 1975 - 2541 binding ( see 
  a ) . integrin binding to the ibs2 region has been reported previously , and we 
  have confirmed this by pulldown and elisa assays . we also show for the first 
  time that ibs2 binds to all -integrin tails with comparable affinity , 
  and moreover an affinity that is only 2-fold weaker than binding by the 
  talin ferm domain . however , our studies contrast with earlier reports 
  ( 24 , 
  25 , 
  44 ) by showing that tight 
  binding to integrins requires both five - helix bundles . moreover , only the 
  two - domain ibs2 polypeptide localized strongly to fas in vinculin - null cells , 
  whereas the individual five - helix bundles showed a diffuse cytoplasmic 
  distribution ( vinculin - null cells were used because ibs2-a contains a 
  vinculin - binding site ) . because the structures of the individual bundles 
  appear not to be significantly influenced by the presence of the other bundle 
  ( as judged by nmr ) we showed that this was indeed the case by using dsc , which 
  clearly indicated binding of integrin tails to both bundles . we considered the 
  possibility that integrin binds to residues at the interface between the two 
  domains , but as these are not conserved this seems unlikely . figure 8.dsc analysis shows binding of various talin rod domains to 
  -integrin tail peptides in solution . dsc analysis of talin 
  ( a and d ) 1974 - 2293 , ( b and e ) 1974 - 2140 
  ( ibs2-a ) , and ( c and f ) 2137 - 2293 ( ibs2-b ) in the presence 
  of 47-mer 1a - integrin peptide ( a - c ) and 25-mer 
  1a - integrin membrane proximal peptide ( d - f ) . the concentration 
  of the talin constructs was 0.7 mg / ml . dsc analysis shows binding of various talin rod domains to 
  -integrin tail peptides in solution . dsc analysis of talin 
  ( a and d ) 1974 - 2293 , ( b and e ) 1974 - 2140 
  ( ibs2-a ) , and ( c and f ) 2137 - 2293 ( ibs2-b ) in the presence 
  of 47-mer 1a - integrin peptide ( a - c ) and 25-mer 
  1a - integrin membrane proximal peptide ( d - f ) . the concentration 
  of the talin constructs was 0.7 mg / ml . using blot overlays and dsc experiments , we mapped the major determinants 
  of ibs2 binding to the membrane proximal ( 23 residue ) region of the 
  -integrin tails , although more membrane - distal regions also contribute 
  to strong binding . we further found that alanine substitution of acidic 
  residues in -integrin tails consistently and uniquely increased ibs2 
  binding , while substitution of basic residues had the opposite effect , 
  suggesting that the interaction has a strong electrostatic component . ( 44 ) has also 
  presented evidence that electrostatic interactions are important in ibs2 
  binding to -integrin tails but concluded that two acidic residues in the 
  3-integrin tail , glu-726 and glu-733 were involved in binding to 
  lys-2085 and lys-2089 on the surface of talin helix 50 in ibs2-a . it should be 
  noted , however , that their experiments employed a fragment of talin 
  ( 1843 - 2108 ) that includes part of ibs2-a but lacks the last -helix . our studies also differ in significant respects from those of tremuth 
  et al . ( 24 ) , who 
  mapped the integrin - binding site to talin residues 1984 - 2113 . our crystal 
  structure shows that this fragment comprises part of ibs2-a only and lacks 
  part of the first helix and the whole of helix 5 , raising significant concerns 
  over its structural integrity and utility in binding studies . however , our dsc 
  experiments show that integrin - binding destabilizes both ibs2-a and ibs2-b 
  bundles , raising the possibility that the 1984 - 2113 construct favors integrin 
  binding precisely , because the structure is destabilized . if so , this would be 
  strongly reminiscent of our studies of vinculin binding , where the talin 
  bundles must unfold to engage the vinculin head or f - actin , a process that is 
  favored by truncated or mutant variants of the talin bundle with altered 
  stability ( 16 , 
  42 ) . indeed , moes et 
  al . ( 25 ) showed that 
  gst-3-integrin tails bound to an immobilized talin peptide corresponding 
  to a single helix ( helix 50 , residues 2077 - 2099 ) in the talin ibs2-a bundle . the significance of these studies employing subdomain fragments remains to be 
  determined . in an attempt to characterize the interaction between talin ibs2 and 
  -integrin tails in more detail , we performed nmr experiments using 
  unlabeled 3-integrin peptides and n - labeled talin 1974 - 2293 
  ( in collaboration with s. lorenz , n. anthis , and i. d. campbell ) . unfortunately , the complex precipitated ( both the integrin peptide and talin 
  polypeptide could be detected in the precipitate by sds - page ) . precipitation 
  was seen with the high affinity fragments , full - length 3-integrin tail 
  ( residues 716 - 762 ) and the membrane proximal peptide ( residues 716 - 740 ) , but 
  not with weakly binding membrane - distal peptides ( residues 736 - 749 and 
  744 - 762 ) . precipitation at the high concentrations employed for nmr studies is 
  consistent with the dsc experiments suggesting that ibs2 partially unfolds 
  upon binding integrin . in vivo , it is conceivable that conformational 
  changes induced by integrin binding to ibs2 are stabilized by association with 
  the plasma membrane , which is in close proximity , and our demonstration that 
  ibs2 binds acidic phospholipids in vitro may be relevant here . although speculative at this point , destabilization of the ibs2 bundle might 
  also lead to exposure of the vinculin - binding site present in helix 50 ( or 
  vice versa ) , resulting in vinculin binding and stabilization of the 
  focal adhesion complex . talin is thought to exist in an inactive form in which the integrin - binding 
  sites are masked , and the recent studies of goksoy et al . ( 45 ) have provided the first 
  insights into the structural basis for such autoinhibition . thus , they have 
  shown that the talin f3 ferm subdomain binds to residues 1654 - 2344 in the 
  talin rod , and that this interaction masks the binding site for the membrane 
  proximal helix of the -integrin cytoplasmic domain in f3 . the above 
  region of the talin rod overlaps with ibs2 , and it will be interesting to see 
  whether binding of f3 to the rod also masks ibs2 , i.e. the two 
  integrin - binding sites may be co - regulated .
the adaptor protein talin serves both to activate the integrin family of 
  cell adhesion molecules and to couple integrins to the actin cytoskeleton . 
 integrin activation has been shown to involve binding of the talin ferm domain 
  to membrane proximal sequences in the cytoplasmic domain of the integrin 
  -subunit . 
 however , a second integrin - binding site ( ibs2 ) has been 
  identified near the c - terminal end of the talin rod . here 
 we report the 
  crystal structure of ibs2 ( residues 1974 - 2293 ) , which comprises two five - helix 
  bundles ,  ibs2-a 
 ( 1974 - 2139 ) and  ibs2-b  
  ( 2140 - 2293 ) , connected by a continuous helix with a distinct kink at its 
  center that is stabilized by side - chain h - bonding . 
 solution studies using 
  small angle x - ray scattering and nmr point to a fairly flexible quaternary 
  organization . using pull - down and enzyme - linked immunosorbent assays , we 
  demonstrate that integrin binding requires both ibs2 domains , as does binding 
  to acidic phospholipids and robust targeting to focal adhesions . 
 we have 
  defined the membrane proximal region of the integrin cytoplasmic domain as the 
  major binding region , although more membrane distal regions are also required 
  for strong binding . 
 alanine - scanning mutagenesis points to an important 
  electrostatic component to binding . 
 thermal unfolding experiments show that 
  integrin binding induces conformational changes in the ibs2 module , which we 
  speculate are linked to vinculin and membrane binding .