the 
 human tyrosine kinase receptor family is comprised of trka , 
 trkb , and trkc .
trka and trkb have become targets for drug discovery 
 for treating conditions ranging from pain and cancer to schizophrenia 
 and alzheimer s disease .
here we focus on the 
 design and structure determination of a stable construct of the extracellular 
 trkaig2 domain suitable for use in nmr to provide binding site information 
 in drug discovery projects . in the periphery , trka mediates 
 nociceptive sensitization
the trk receptors are comprised of a 
 leucine / cysteine rich domain and two immunoglobulin - like domains , 
 ig1 and ig2 , in the extracellular region linked to an intracellular 
 kinase domain by a single membrane - spanning helix .
the ig2 domain 
 is proximal to the cell membrane and provides the ngf binding site 
 as was first indicated by urfer et al . and later shown by x - ray crystallography .
ngf 
 is a homodimeric protein presenting two trka binding sites on opposite 
 faces of the dimer .
the binding of ngf brings together two trka receptors 
 triggering autophosphorylation of their intracellular kinase domains . 

autophosphorylation provides binding sites for proteins involved in 
 downstream signaling in the phosphatidylinositol-3 ( pi3 ) -kinase , 
 mitogen - activated kinase / extracellular - signal - regulated kinase ( map 
 kinase / erk ) , and phospholipase c- ( plc- ) pathways .
consequentially , ngf activation of trka modulates the activity of 
 both ligand and voltage - gated ion channels involved in nociception 
 via the following mechanisms . under resting conditions
, the ligand - gated 
 transient receptor potential cation channel vanilloid subfamily member 
 1 ( trpv1 ) is constitutively inhibited by phosphoinositol 4,5-bisphosphate 
 ( pip2 ) on nociceptors .
the activation of plc- downstream of 
 trka activation by ngf relieves this constitutive inhibition by hydrolyzing 
 pip2 to inositol 1,4,5-trisphosphate ( ip3 ) and diacylglycerol .
pain signaling is enhanced by the release 
 of brain derived neurotrophic factor ( bdnf ) , substance p , and other 
 peptides which are transported to the dorsal root ganglion ( drg ) , 
 subsequently promoting central pain perception .
the activation of trka by ngf thus initiates the chronic 
 pain phenotype by increasing nociceptor sensitivity to further stimulus . during the sensitization process
, trka expression 
 is itself up - regulated on nociceptors , as are the ion channels nav1.8 , 
 p2xy , and trpv1 .
however , within 
 the brain , trka function is fundamental to memory 
 formation and learning .
cholinergic cells 
 of the basal forebrain extend axons into the hippocampus and cerebral 
 cortex and depend on the supply of ngf delivered by axonal retrograde 
 transport .
activation of trka by ngf 
 triggers the aforementioned signaling cascades to provide support 
 for neurite growth and repair and maintenance of effective connectivity 
 between the cholinergic basal forebrain , hippocampus , and the cortex 
 for the formation of new memories .
early in the progression of alzheimer s 
 disease , trka / ngf signaling in the cholinergic cells of the basal 
 forebrain is compromised , the cells undergo axonal withdrawal , and 
 as a result , brain regions no longer communicate efficiently .
trka 
 is a validated target for pain therapeutics in both animal 
 models and in the clinic .
ngf based therapeutics in animal models 
 of pain including the ngf binding domain on trka ( trkaig2 ) and anti - ngf antibodies including tanezumab have successfully provided relief from acute and chronic pain states 
 in clinical trials . however , several 
 antibody therapies have had serious side effects which may arise from the high affinity of the antibody / target 
 interaction combined with the very long half - life of the ngf antibody , 
 making bioavailability hard to predict and hence control by dosing . identifying small molecule therapeutics targeted to the extracellular 
 trka domain to disrupt the trka / ngf protein 
protein interaction 
 provides a major challenge , and high - throughput screens have failed 
 to produce small molecule antagonists to this target .
kinase inhibitors 
 specific for trka are difficult to achieve as the intracellular kinase 
 domains are highly conserved between the trk receptors : a to b , 75% 
 identity , a to c , 76% identity , and b to c , 82% identity and all have 
 almost identical atp binding sites .
targeting the extracellular 
 ngf binding domain of trka , the trkaig2 
 domain , provides potential advantages for two main reasons : influencing 
 the extracellular ligand binding interaction represents the preamplification 
 stage in the signaling cascades where nature  fine - tunes  
 receptor - mediated events ; the neurotrophin binding domains share lower 
 sequence identity between trk receptors : a to b , 46% , a to c , 41% , 
 and b to c , 47% , making specific targeting more readily achievable . 

compounds that can prevent ngf - mediated activation of trka on peripheral 
 nociceptors will be useful in the management of pain and those capable 
 of acting centrally to augment ngf - mediated activation of trka could 
 be developed as alzheimer s therapeutics to support the cholinergic 
 system .
to develop trkaig2 domain binders , access to reproducible 
 structural 
 information is required to confirm the compound binding site .
crystallography 
 has proven to be problematic because at high concentrations the isolated 
 ig2 domain forms -strand - swapped dimers ( 1wwa.pdb ) in the absence 
 of its cognate ligand ngf and is thus biologically inactive .
strand - swapping occludes not only the ngf binding 
 site but also the compound binding sites targeted .
we describe 
 the design and validation of a trkaig2-nmr construct 
 in which we have stabilized the native fold ( not strand - swapped ) and 
 retained biological activity . here
we use solution nmr to determine 
 the three - dimensional structure of the protein and validate the use 
 of the construct using ngf and a small molecule ( amitriptyline ) in 
 both nmr binding experiments and functional assays .
this approach 
 should greatly facilitate the development of small molecule leads 
 for modulating the activity of trka and related receptors .
initially , 
 we selected an in - house construct spanning the second ig domain of 
 trka that we refer to as  wild - type  trkaig2 ( trkaig2-wt ) 
 ( figure 1a ) .
this construct has been used successfully 
 to sequester ngf and abrogate pain in a number of in vivo models and was therefore used for initial nmr studies .
a n - labeled 
 sample of the trkaig2-wt construct was prepared and a two - dimensional h 
this spectrum displayed many unfavorable features 
 for structural and ligand - binding assays , including overlap and heterogeneity 
 of signal intensity ( e.g. , a mixture of intense and weak broadened 
 cross - peaks ) .
trkaig2-wt 
 domain , the first - generation engineered cysteine mutant ( trkaig2-ds1 ) , 
 the final construct produced for solution nmr studies ( trkaig2-nmr ) , 
 and the construct used in x - ray crystallographic studies of the strand - swapped 
 dimer ( trkaig2-xt1 ) .
the amide peak dispersal indicates that the protein is not a homogeneous 
 population or that some of the structure is disordered . to engineer a construct for use in the absence 
 of ngf , we re - examined 
 known crystal structures of these proteins .
the x - ray crystallographic 
 study of the wild - type single trka - ig2 domain ( figure 2a ) bound to ngf previously revealed two molecules of the trka - ig2 
 domain bound to a central ngf dimer .
an 
 extensive interface was observed between ngf and the individual trka - ig2 
 domains , but there were no direct contacts between the trka - ig2 domains 
 themselves . when studied in isolation ,
however , the ig2 domains from 
 trka , trkb , and trkc have all been shown to form strand - swapped dimers 
 in solved crystal structures ( figure 2b ) , an 
 association that occludes the interaction site on trka for ngf . therefore
, 
 at high concentrations , the formation of oligomeric species in addition to unstructured regions might explain 
 the poor nmr characteristics of the trkaig2-wt construct .
figure 2c shows an overlay of a single trka - ig2 domain chain 
 from the strand - swapped crystal structure 1he7.pdb with a single chain from the crystal 
 structure 1www.pdb in complex with ngf .
the strand - swapped structure of trka - ig2 
 ( residues 285413 ) , studied by robertson et al . , revealed a 
 stable core structure extending to p382 after which no electron density 
 was observed .
the c - terminal was presumed 
 to be flexible and might not therefore be ideal for solution - state 
 nmr studies .
therefore , the c - terminal was truncated to end in the 
 residues dnpf ( 383 ) ( figure 1a ) . to prevent 
 -strands swapping between adjacent monomers at high concentrations , 
 an additional disulfide bond was also introduced to act as an intramolecular 
 staple between -strand 1 ( p285c ) and the -hairpin between 
 strands 6 and 7 ( f367c , figure 1a ) to yield 
 our first - generation construct trkaig2-ds1 .
the disulfide bridge was 
 modeled onto chain x of trka from the crystal structure 1www.pdb and then energy 
 minimized using discover 2.98 ( accelrys ) ( figure 2d ) .
these residues were chosen because they were suitably 
 distant from the ngf binding face of the protein and therefore less 
 likely to influence the binding of compounds to the target site .
p285 
 and f367 were already almost the optimal distance apart required for 
 disulfide bond formation . in the crystal structure , their side chains 
 were oriented toward each other so that disulfide formation between 
 two cysteines at these positions would be predicted to cause minimal 
 distortion to the overall fold .
importantly , these residues fulfilled 
 another necessary criterion predicted 
 to produce a hyper - stable native state , namely they were not involved 
 in the hydrogen bonding pattern of the -sheet .
comparison of x - ray crystallographic 
 structures and modeled trka - ig2 
 in free forms and complexed with ngf .
( a ) crystal structure of the 
 complex between ngf and trka - ig2 ( 1www.pdb ) with 
 two molecules of trka - ig2 shown in magenta in binding to an ngf dimer 
 ( slate ) .
( b ) crystal structure of two molecules of trka - ig2 ( red and 
 light - orange ) in the absence of ngf forming a strand - swapped dimer 
 ( 1wwa.pdb ) .
( c ) superposition of the x - ray structure of a 
 further strand - swapped dimer of trkaig2 ( pdb code 1he7 ) ( cyan ) with the position of p285 and f367 highlighted . 

the structure of modeled domain 5 ( green ) is shown incorporating the 
 p285c and f367c mutations and the new disulfide bond after energy 
 minimization .
the n - terminal strand in the modeled 
 construct is highlighted with * , and the point at which the non- and 
 strand - swapped dimers diverge is indicated by * * .
( d ) similarly , superposition 
 of the minimized disulfide bridged construct shows close structural 
 similarity with the packing of the n - terminal -strand in the 
 ngf bound crystal structure ( 1www.pdb ) .
comparison of the predicted model of trkaig2-ds1 with the 
 crystal 
 structure of the strand - swapped dimer ( figure 2c ) shows the expected drastic change in the packing 
 of the n - terminal strand but minimal perturbation to the remainder 
 of the protein .
similarly , comparison of the predicted model with 
 the nonstrand - swapped crystal structure of the trka - ig2 domain in 
 complex with ngf shows only small perturbations in the region of the 
 disulfide bridge which were unlikely to influence the more distant 
 ngf binding site ( figure 2d ) .
trkaig2-ds1 
 was expressed and purified but yields of protein were 
 low , so a further construct which included an additional 5 n - terminal 
 aspartates ( trkaig2-nmr ) was produced ( figure 1a ) .
the hypothesis was that an increased overall negative charge 
 ( pi with aspartates 4.52 ) might reduce nonspecific 
 association during refolding and reduce the potential for inappropriate 
 disulfide bonding during the refold by not having the cysteine at 
 position one in the sequence .
yields of the second - generation 
 protein , termed trkaig2-nmr , were 
 improved as the proportion of protein aggregating on refold was significantly 
 reduced .
biological activity 
 was determined through the ability of trka constructs to bind and 
 sequester ngf .
the trkaig2-nmr reduced cell survival ( figure 3a ) with an ec50 of 2.1 m compared 
 with trkaig2-wt ec50 0.48 m ) .
trkaig2-nmr inhibition 
 of ngf - induced neurite outgrowth on pc12 cells ( figure 3b ) gave an ec50 of 1.59 m .
this construct 
 also sequestered radiolabeled ngf in competition assays with an approximate 
 ic50 of 5 m on hek cells expressing human trka ( figure 3b , inset ) .
a maldi - tof spectrum confirmed 
 the presence of a single monomeric species and no disulfide - linked 
 dimer was detected and native gel electrophoresis showed a single 
 band ( supporting information , figure s1 ) .
analytical ultracentrifugation of the trkaig2-nmr construct confirmed 
 that the protein was monomeric under the conditions used for the nmr 
 studies ( supporting information , figure s1 ) , and gel filtration showed a single symmetrical peak ( figure 3c ) .
taken together , these results indicate the construct 
 was a monomeric homogeneous fold . functional and biophysical characterization 
 of trkaig2-nmr compared 
 to trkaig2-wt .
( a ) trkaig2-nmr and trkaig2-wt effect on ngf - dependent 
 cell proliferation in pc12 cells .
both the trkaig2-wt ( closed circles , 
 ec50 0.48 m ) and the trkaig2-nmr ( open circles , 
 ec50 2.1 m ) forms were able to sequester ngf and 
 prevent cell proliferation in a dose - dependent manner .
( b ) the effect 
 of trkaig2-nmr on ngf - mediated neurite outgrowth in pc12 cells : 0.04 
 nm ngf was added to pc12 cells in addition to a range of concentrations 
 of n labeled trkaig2 .
inset : n - labeled trkaig2-nmr mediated sequestration of i - labeled ngf in a competition assay against full - length human trka 
 receptors expressed on hek cells , with an approximate ic50 of 2 m .
trkaig2-nmr and mouse ngf were applied to a gel filtration s75 column , 
 separately and as a 1:1 ratio complex of two monomers of trkaig2-nmr 
 with one dimer of ngf .
the trkaig2-nmr / ngf complex ( mwt 51 kda ) was 
 shown to run at 57 kda as estimated by a calibration curve . alone
, 
 trkaig2-nmr ( mwt of monomer is 11.9 kda ) ran at 17 kda ; ngf ( 27 kda 
 dimer ) ran at 18.8 kda .
n hsqc 
 spectra of trkaig2-nmr , showing vastly improved homogeneity of peak 
 intensity and line shape .
n hsqc was recorded on 
 a n - labeled sample of trkaig2-nmr ( figure 3d ) and showed distinct improvements to both peak dispersion 
 and uniformity of peak height .
this construct also had the advantage 
 of being stable for at least three months at 25 c and provided 
 reproducible hsqc spectra between preparations .
the assignment 
 of the h , c , and n resonances 
 of trkaig2 were obtained using a c , n labeled 
 sample of trkaig2-nmr and triple resonance experiments 
 recorded at 600 mhz .
overall , 98% of the backbone resonances could 
 be assigned , with missing residues being located in the n - terminal 
 asp - rich region ; 94% of the side - chain resonances could be assigned . 

the solution structure was determined using a total 
 of 182 dihedral angles , 46 hydrogen bonds , and 2053 noes derived from 
 3d n - edited noesy - hsqc , c aromatic hsqc - noesy , 
 and simultaneous n / c - edited noesy spectra . 

the final twenty structures chosen to represent the ensemble of nmr 
 structures ( figure 4a ) were consistent with 
 both experimental data and standard covalent geometry , displaying 
 no violations greater than 0.5  for distance restraints or 5 
 for dihedral angles .
the averaged structure for the ensemble highlights 
 the position of the disulfide staple ( figure 4b ) .
details of the final set of structural restraints and their violations 
 within the final ensemble are listed in table 1 .
( b ) the closest 
 to the geometric average solution structure with the position of the 
 disulfide staple shown between c285 and c367 .
( c ) an overlay of chain 
 x from the crystal structure of the trkaig2/ngf complex ( magenta , 1www.pdb ) and the closest to the geometric average nmr 
 model of the trkaig2-nmr ( green ) construct showing that the disulfide 
 bridge has not disrupted the global protein fold .
the core of trkaig2-nmr is well - defined , with a backbone 
 rmsd from 
 the mean over the regular secondary structure elements of 0.38  
 for the ensemble of structures .
as expected , the solution structure 
 forms an ig - like fold consisting of a -sandwich formed by two 
 -sheets .
the structure is highly similar to the 2.2  
 crystal structure of trka in complex with ngf solved at ph 5.0 ( 1www.pdb ) with a backbone rmsd over the secondary structure elements 
 of 0.68  ( profit v2.5 ) ( figure 4c ) .
as 
 expected , the introduction of a disulfide bond between p285 and f367 
 to help stabilize the protein does not affect the overall structure .
minor differences in the solution and crystal structures are observed 
 in the loop regions due to ph or conformational changes .
the complex 
 was crystallized by hanging drop in 1:1 protein solution : hampton screen . 

the protein solution was 10 mg ml complex ( trka 
 and ngf ) in 0.1 m nacl , 0.1 m bicine , ph 8.5 , the hampton screen condition 
 comprised of 24% peg 3350 , 0.1 m citric acid , ph 5.0 ( 1www.pdb ) .
buffer conditions 
 for the nmr were 100 mm sodium phosphate ph 6.9 and 10 mm nacl .
no 
 evidence of dimer formation was observed in the nmr experiments , and 
 testing the calculated models against a possible dimeric stoichiometry 
 gave no improvement in the fit to the noe data or minimized energies .
protein interaction in solution 
 under conditions used for nmr with the trka receptor s cognate 
 ligand , the cytokine ngf ( from mouse submaxillary salivary glands ) .
ngf readily adsorbs onto many surfaces , but reversible acid denaturation reduces this 
 propensity .
a single - shot nmr assay was recorded to ensure that no ph 
 perturbation occurred when the complex was formed as 650 g 
 of ngf ( preadjusted to ph 6.9 ) was added to a solution of n - trkaig2-nmr to approach a 1:1 stoichiometry .
n hsqc spectra before ( black ) and after ( red ) the addition 
 of a stoichiometric concentration of ngf .
a number of residues show 
 either amide chemical shift perturbations ( csps ) or are significantly 
 line broadened in the presence of ngf ( figure 5b ) .
these perturbed residues were mapped onto the crystal structure 
 of the trkaig2 domain bound to ngf and are shown in figure 5c , d .
although the csps are < 0.15 ppm , the predominant spectral changes cluster around 
 the groove formed by strands 2 , 4 , and 5 that 
 binds the n - terminal helix of ngf with several additional residues 
 in the loop connecting strands 5 and 6 .
in particular , residues which are almost completely line 
 broadened ( shown as green bars in figure 5b ) 
 form a patch centered in the binding groove which would contact the 
 helix ( figure 5c ) .
this n - terminal helix is 
 unstructured when ngf is crystallized in the free form ( 1bet.pdb ) , and electron 
 density is only visible for this region when ngf is in complex with 
 the trkaig2 domain ( 1www.pdb ) and makes an energetically important contribution to the binding 
 energy of the trkaig2/ngf interaction .
we note however that the observed peak broadening is not as pronounced 
 as expected if a large , stable complex was formed with ngf ( kd low micromolar ) , suggesting that not all of 
 the ngf was available in solution , perhaps due to binding to the glass 
 of the nmr tube and the trkaig2-nmr remains well in excess .
n hsqc spectra of trkaig2-nmr construct before 
 ( black ) and after ( red ) the addition of stoichiometrically equivalent 
 quantity of ngf .
resonances that show chemical shift perturbations 
 ( csps ) or appear significantly broadened have been annotated .
( b ) 
 nh between free trkaig2-nmr and ngf present 
 ( orange ) , where the height is proportional to the difference in ppm . 

the majority of the residues that show csps or line broadening 
 interact with the n - terminus of ngf that forms a helix on binding 
 the trkaig2 domain .
( c ) surface representation of trkaig2-nmr ( dark - gray ) 
 and ngf ( cyan ) with csps and line broadening shaded orange and exchange - broadened 
 peaks only shaded green .
( d ) ngf ( cyan ) is depicted in secondary structure 
 as a ribbon in complex with one trkaig2-nmr ( green ) , with residues 
 corresponding to the peaks shifted in the h 
next 
 we tested whether we could observe an interaction of trkaig2-nmr with 
 a small molecule ligand .
the national institute for health and clinical 
 excellence ( nice ) supports the use of amitriptyline for the management 
 of neuropathic pain , and therefore amitriptyline was one of a number 
 of similar compounds tested in vitro by us and found to displace i - ngf from human trka expressed on hek cells ( see below ) . 

addition 
 of amitriptyline to trkaig2-nmr ( 100 m ) gave over 20 observable 
 amide csps with five > 0.2 ( figure 6b ) .
the csps observed saturate with increasing amitriptyline 
 concentration , allowing the extraction of the kd values for four residues ( t292 kd 1.8 mm  0.2 mm , v305 kd 2.1 mm 
  0.4 mm , c345 kd 2.1 mm  0.4 
 mm , f303 kd 2.2 mm  0.1 mm ) ( figure 6c ) .
the difference in kd observed for amitriptyline binding to trkaig2-nmr versus the ic50 observed with full - length trka indicates some loss of affinity 
 when utilizing this engineered single domain .
although the kd is only in the mm range , this low molecular 
 weight species ( which lies within the < 300 da fragment classification ) 
 shows 13 common perturbed residues ( csp or line broadening ) with ngf 
 ( figures 5a and 6a , b ) 
 and include l290 , t292 , g344 , c345 , l346 , and l348 .
these residues 
 again map to the groove on the trkaig2 domain formed by strands 2 , 
 4 , and 5 and interconnecting loops where the n - terminal 
 helix of ngf binds (  site 1  ) .
several larger csps , 
 however ( e.g. , s304 and v305 ) , are only significantly perturbed by 
 amitriptyline and arise from residues that lie within a narrower groove 
 formed between stands 1 and 2 adjacent to the ngf helix 
 binding site (  site 2  ) . to further explore the bound 
 conformation of amitriptyline
, the compound was initially positioned 
 by hand guided by the clustered csps at the known ngf binding site . 

amitriptyline was then docked into the binding site on the trkaig2 
 domain using bristol university docking engine ( bude ) and poses judged against fits to the observed h csps .
seven of the top 10 poses from 
 bude docking with best predicted binding energies were selected ( see supporting information , figure s4 and table s1 ) . a representative conformer from these models is shown in figure 6d and covers both site 1 and site 2 defined by the 
 csps .
amitriptyline binding in this position would be expected to 
 interfere with ngf binding at site 1 as illustrated but does not completely 
 occlude the ngf n - terminal helix binding groove ( figure 6d ) .
the model also shows that amitriptyline binding extends 
 somewhat beyond the binding site of the n - terminus of ngf to site 
 2 .
this may have implications for the design of further small molecule 
 binders targeting trka .
( b ) nh between free and amitriptyline bound trkaig2-nmr ( blue ) , 
 where the height is proportional to the difference in ppm .
also shown 
 are the csps for the interaction with ngf for comparison ( orange ) . 

the negative points again indicate that line broadening was observed 
 but no significant csp .
( c ) titration with amitriptyline provides 
 a saturable signal . f303 and g344 show amide peaks shift with titrated 
 amitriptyline , and both the change in peak positions and the resulting 
 chemical shift change versus amitriptyline curves are shown as insets . 

( d ) amitriptyline docked into the groove identified by peak shifts 
 and inset surface representation of amitriptyline docked into trkaig2-nmr . 

the n - terminal helix of ngf which is partially blocked ( site 1 ) by 
 amitriptyline binding is shown in blue . both h291 and h343 show csps . 

( e ) amitriptyline competes with radiolabeled ngf for binding to the 
 full - length trka receptor expressed on hek cells with ic50  60 m .
( f ) amitriptyline antagonizes downstream signaling 
 ( phosphorylation of erk 42/44 ) normally triggered by the ngf / trka 
 interaction in automated immunofluorescence ( incell ) assays ( ge healthcare ) 
 with an ec50  86 m .
dose related displacement 
 of i - ngf by amitriptyline was assessed using a competition 
 assay ( see materials and methods ) .
the acid 
 salt of amitriptyline is freely soluble in water ; therefore , to avoid 
 any unforeseen effects , dimethyl sulfoxide ( dmso ) was not used in 
 any assays or nmr experiments .
our results showed that amitriptyline 
 competes with radiolabeled ngf for binding to human trka with an ic50 60 m ( figure 6e ) .
automated 
 immunofluorescence ( incell ) analysis also shows that amitriptyline 
 inhibits the phosphoerk signal induced downstream of trka activation 
 by 0.5 nm ngf on hek cells expressing the full - length human trka receptor 
 with an ec50 of approximately 86 m ( figure 6f ) .
evidence of competition with respect to ngf 
 on cells expressing human trka corroborates the observed peak shifts 
 on the trka - ig2-nmr , both indicating binding site overlap between 
 amitriptyline and ngf .
the trka / ngf interaction 
 is an important and well - validated target 
 for pain intervention . by modifying the trkaig2 domain ,
we have produced 
 a valuable tool for assessing the binding of compounds to a site on 
 trka in order to inhibit the binding of ngf .
the main problems we 
 have overcome were protein flexibility and strand - swapping at high 
 concentrations .
protein flexibility was reduced by truncating the 
 c - terminus to exclude residues 384413 , and strand - swapping 
 was abolished by the addition of a disulfide bond to  staple  

the addition of five 
 n - terminal aspartic acid residues further improved monomer yield in 
 the refold .
overall , these mutations provided a stable construct , 
 and the three - dimensional solution nmr structure has an identical 
 fold to that of the trkaig2 domain in its native conformation bound 
 to its cognate ligand ngf ( pdb code 1www ) .
the hsqc spectra are robustly reproducible 
 between batches , facilitating the rapid determination of the weakly 
 binding compound amitriptyline .
because of the construct being an 
 excised domain , we observed a reduction in binding affinity versus 
 the full - length receptor , however , this successfully located the binding 
 site and generated csp data of sufficient quality to be used as additional 
 docking restraints by bude .
therefore , we suggest that in conjunction 
 with traditional binding and functional assays trkaig2-nmr will be 
 a useful tool in the hit to lead optimization of therapeutics as antagonists 
 for pain or agonist / modulators for cholinergic support in alzheimer s 
 disease .
the residues chosen to 
 form a disulfide staple were mutated using chain x from the crystal 
 structure 1www.pdb in insightii 2005 .
the complex was soaked with a 10  layer 
 of water and then energy - minimized using discover 2.98 ( accelrys inc .
subsequently , all atoms were gradually allowed freedom 
 to move , enabling backbone and side chain flexibility as the system 
 relaxed .
images were generated using a combination of chimera ( university 
 of california san francisco ) , gimp ( gnu image manipulation program , http://www.gimp.org/ ) , and powerpoint ( microsoft ) .
guided by the amide shifts induced 
 by the binding of amitriptyline to the trkaig2-nmr construct , the 
 compound was maneuvered into position by hand using insightii .
the 
 complex was soaked with a 10  layer of water and then energy - minimized 
 as described above . to verify this position ,
the docking software 
 bude was used to dock amitriptyline into the center of the binding 
 area highlighted by the peak shifts on hsqc .
fourteen different conformers 
 of amitriptyline were produced and then allowed to rotate around 360 
 and translate by 12  in x , y , and z ( a total of 8.25 million poses were sampled ) . 

poses were initially ranked according to their bude predicted binding 
 energy and subsequently by the minimum distance between any amitriptyline 
 atom and the predominant amide , hn ( proton ) csps ( t292 , f303 , s304 , 
 v305 , h343 , g344 , c345 , and l346 ) achieved by amitriptyline binding .
the trkaig2-ds1 construct represents residues 
 285383 comprising the ngf binding domain of the human trka 
 receptor ( p04629 ) . a full length trkaig2 domain 7.2cis pet24a ( + ) 
 plasmid was used as a pcr template ( residues 285413 ) .
this 
 template included p285c and p367c mutations and had been engineered 
 to reduce mrna secondary structure by the incorporation of silent 
 mutations ( residues 286290 ) to favor ribosome binding .
these 
 silent mutations facilitated the eventual production of recombinant 
 trkaig2-nmr within an escherichia coli expression system .
amplification of trkaig2-ds1 
 from trkaig2 d57.2cis 
 pet24a(+ ) by pcr with pfu polymerase ( promega ) using primers ( ggaattccatatggatgacgatgacgatgttagcttttgtgcttcagtacaatta ) 
 and ( ccgctcgagttatcagaaagggttgtccatgaaggcagccatg ) produced a 
 pcr product that was subcloned into the ndei and xhoi cloning sites 
 of pet24a(+ ) vector ( novagen ) and transformed into a nonexpression 
 host xl1 blue ( stratagene ) .
purified mutant construct pet24a(+)trkaig2-ds1 
 was checked by sequencing and then transformed into a bl21(de3 ) e. coli ( novagen ) expression host .
the penta - asp tagged trkaig2-nmr construct 
 was made by amplification of the trkaig2-nmr from template pet24a(+)trkaig2-ds1 
 by pcr with pfu polymerase ( promega ) using the following primers : 
 forward primer including an ndei site ( ggaattccatatggatgacgatgacgatgttagcttttgtgcttcagtacaatta ) 
 and the reverse primer incorporating an xhoi site ( ccgctcgagttatcagaaagggttgtccatgaaggcagccatg ) .
this corresponds to the protein sequence : mdddddvsfcasvqlhtavemhhwcipfsvdgqpapslrwlfngsvlnetsfifteflepaanetvrhgclrlnqpthvnngnytllaanpcgqasasimaafmdnpf . to produce n or c , n - labeled trkaig2-nmr , bl21(de3)trkaig2-nmr , 

cells were grown overnight in 50 ml of m9 minimal media ( 42 mm na2hpo4 , 22 mm kh2po4 , 9 mm 
 nacl , 45 mm cacl2 , 2 mm mgso4 , 2 mg / ml thiamine 
 ( sigma ) ) , containing 0.2% d - glucose c ( for c preparations only ) , ( c6 ) ( cambridge isotope laboratories ) , 
 10 mm nh4cl , 99% ( cambridge isotope laboratories ) 
 and 0.5 mg / ml of kanamycin ( sigma ) at 37 c .
the cells were then 
 diluted 1:10 in fresh media and grown at 37 c to an od595 = 0.6 .
isopropyl - d - thiogalactoside ( iptg ; sigma ) was then 
 added to 1 mm , and the cells were grown overnight .
unlabeled protein 
 was produced using the same protocol as outlined here except that 
 minimal media with nutrient additives were replaced by luria 
bertani 
 ( lb ) culture media throughout . cells ( 8 g wet weight ) were harvested 
 by centrifugation at 8000 rcf at 4 c for 15 min and resuspended 
 into 50 ml of ice - cold , sterile 10% ( v / v ) glycerol .
pellets were stored 
 as 25 ml aliquots and frozen at 80 c until required . 

briefly , each bacterial pellet was disrupted using a french - press 
 and resuspended in 200 ml of ice - cold resuspension buffer ( 20 mm tris 
 ph 8.5 , 10 mm edta ) containing 100 mm nacl and centrifuged at 9000 
 rcf at 4 c for 60 min .
three further rounds of resuspension 
 were then carried out with centrifugation at 9000 rcf at 4 c 
 for 30 min .
each pellet was resuspended in 100 ml of resuspension 
 buffer with sequential additions of 1 m nacl , 1% ( v / v ) triton x-100 , 
 and 100 mm nacl .
each inclusion body pellet was resuspended in 50 ml of a solubilization 
 buffer comprised of 8 m urea , 40 mm tris ph 8.2 , 100 mm nacl , and 
 1 mm -mercaptoethanol ( bme ) and rocked on a platform for 3 
 h at room temperature .
solubilized trkaig2-nmr was collected as supernatant 
 by centrifugation at 9000 rcf for 1 h at 10 c .
protein concentrations 
 of soluble trkaig2-nmr were estimated by uv absorption of aromatic 
 residues at 280 nm ( molar extinction coefficient is 12740 m cm ) and adjusted by dilution in solubilization 
 buffer to 0.1 mg / ml ( 8 m ) .
diluted , soluble trkaig2-nmr 
 was dialyzed 1:20 overnight against a dialysis buffer comprised of 
 20 mm tris at ph 8.2 , containing 50 mm nacl , at 4 c using 28 
 mm diameter dialysis tubing with a molecular weight cutoff ( mwco ) 
 of 3500 da ( medicell international ) .
the partially refolded trkaig2-nmr 
 was again dialyzed 1:20 overnight against the dialysis buffer containing 
 an additional 10 mm nacl .
refolded trkaig2-nmr was centrifuged at 
 8000 rcf at 4 c for 60 min to remove the majority of insoluble 
 protein ; the supernatant was collected and stored on ice .
trkaig2-nmr 
 supernatant was loaded onto a pre - equilibrated 5 ml 
 hitrap q ff column ( amersham bioscience ) at 5 ml / min on an akta fast 
 protein liquid chromatogaph ( fplc ) ( amersham bioscience ) protein purification 
 system .
the column was then re - equilibrated with 5 column volumes 
 of buffer a ( 20 mm tris ph 8.2 , 10 mm nacl ) .
trkaig2-nmr was eluted 
 over a linear gradient consisting of 20 column volumes starting with 
 buffer a and ending with buffer b ( 20 mm tris , ph 8.2 , 1 m nacl ) . 

trkaig2-nmr eluted with a retention 
 time 7.7 min ; fractions were pooled and stored on ice . prior to data 
 acquisition , the pooled fractions of trkaig2-nmr were concentrated 
 to 6.8 mg / ml within 16 mm diameter , 3500 da mwco dialysis tubing ( medicell 
 international ) using polyethylene glycol ( peg ) 20 kda ( sigma ) . the 
 dialysis bag was then transferred to 2000 volume nmr buffer 
 ( 100 mm sodium phosphate ph 6.9 , 10 mm nacl ) and dialyzed overnight 
 at 4 c to exchange buffers and remove any low molecular weight 
 contaminants in the peg .
n hsqc experiments were acquired in 
 nmr buffer , 20 c 
 ( to compare trkaig2-nmr and trkaig2-wt at 100 m concentration ) 
 on a varian inova 600 mhz spectrometer equipped with a room temperature 
 probe .
triple resonance experiment data were acquired at 20 c 
 with a cryoprobe equipped varian vnmrs operating at 600 mhz to assign 
 the backbone and side chain atoms .
n , c 
 noesy - hsqc ( nuclear overhauser effect ( enhancement ) spectroscopy 
heteronuclear 
 single quantum correlation ) experiments were acquired at 600 mhz for 
 distance restraints .
nmr data processing and analysis was performed 
 with nmrpipe and ccpnmr analysis version 
 2.3.1 .
structures 
 were calculated iteratively with cns 1.2 using aria2.3 protocol before 
 being water refined using the recoord protocol .
restraints 
 for the introduced disulfide bond were added once the juxtaposition 
 of the cysteine residues was observed in structure calculations .
final 
 structures were checked with icing ( version r1156 ) ( https://nmr.cmbi.ru.nl/icing/icing.html ) and psvs ( version 1.5 ) ( http://psvs-1_5-dev.nesg.org/ ) . figures and analyses
were produced 
 using pymol ( the pymol molecular graphics system , version 1.5.0.4 , 
 schrdinger , llc ) . and
the ucsf chimera package ( chimera was 
 developed by the resource for biocomputing , visualization , and informatics 
 at the university of california , san francisco ( supported by nigms 
 p41-gm103311 ) ) .
profit v2.5.2 was used 
 to calculate the rmsd between the closest to average nmr structure 
 and chain x of 1www.pdb ( martin , a. c. r. , http://www.bioinf.org.uk/software/profit/ ) .
trkaig2-nmr was typically 100 m 
 in 100 mm sodium phosphate , 10 mm nacl , ph 6.9 .
data was collected 
 on a varian vnmrs 600 mhz nmr spectrometer equipped with a cryogenically 
 cooled triple resonance probe head . for the amitriptyline ( sigma , 
 purity > 98% by tlc ) titration , amitriptyline was added to the following 
 final concentrations : 0 , 0.015 , 0.03 , 0.06 , 0.18 , 0.3 , 0.5 , 1.5 , 3 , 
 and 4 mm . for comparative nmr of the ngf / trkaig2 complex , a stoichiometric 
 equivalent of ngf ( preadjusted to ph 6.9 )
csps ( nh ) were calculated according 
 to eq 1 given below by pellecchia et al.1 auc sedimentation 
 velocity experiments were conducted at 20 c in a beckman optima 
 xl - a analytical ultracentrifuge using an an-60 ti rotor and sedimentation 
 velocity cells equipped with a two - channel aluminum centerpiece and 
 sapphire windows . then 410 l solutions at 80 m protein 
 concentration were loaded in the sample channel , and the reference 
 channel was loaded with 420 l of buffer .
samples were centrifuged 
 at 55000 rcf ( an-60 ti rotor ) , with absorbance scans taken across 
 a radial range of 5.87.3 cm at 3 min intervals to a total 
 of 180 scans .
data were fitted to a continuous c(s ) distribution model 
 using sedfit at a 95% confidence level . the baseline , meniscus , frictional coefficient ( f / f0 ) , and systematic time - invariant and 
 radial - invariant noise were fitted .
the partial specific volume ( v ) 
 for the protein and the buffer densities and viscosities were calculated 
 using sednterp ( http://sednterp.unh.edu/ ) .
the trkaig2-nmr construct 
 has a theoretical pi of 4.52 , therefore 12% polyacrylamide 
 gel electrophoresis was carried under normal ( basic ) conditions with 
 no added sds and no bme in the loading buffer .
trkaig2-nmr and mouse ngf were applied 
 to a gel filtration superdextm 75 ( 10/300 gls in xk16 column with 
 60 cm bed height ) separately and as a 1:1 ratio complex ( 1 mg / ml of 
 each ) .
a calibration column was run in 50 mm phosphate buffer containing 
 150 m nacl at ph 7.4 .
a calibration curve with standard globular 
 proteins ran according to protein molecular weight : dextran blue ( 2000 
 kda ) ( which runs at void volume ) , bovine serum albumin ( bsa ; 67 kda ) , 
 carbonic anhydrase ( 29 kda ) , and cytochrome c ( 12.4 kda ) ; correlation 
 of molecular weight with running volume ( r = 0.98 ) cells expressing 
 the full - length 
 human trka receptors were grown as monolayers in dmem containing 10% 
 fetal calf serum ( fcs ) ( v / v ) , 2 mm glutamine , 10 u / ml penicillin , 
 0.1 mg / ml streptomycin , and 0.5 mg / ml g418 in t75 tissue culture flasks . 

parent hek cells ( lacking expressed trk receptors ) were grown in dmem 
 containing 10% fcs ( v / v ) , 2 mm glutamine , and antibiotics but not 
 g418 . for assays ,
cells were detached from monolayers ( 9095% 
 confluent ) using versene ( phosphate buffered saline ( pbs ) with 1 mm 
 edta ph 8) for 5 min at 37 c and pelleted by centrifugation 
 ( 165 g for 5 min at room temperature ) .
displacement 
 of i - labeled neurotrophin ngf by amitriptyline was assessed 
 in hek n3s cells expressing native human trka .
the cell pellet was 
 washed three times by repeated resuspension and centrifugation , using 
 10 ml of assay buffer ( pbs ph 7.4 ) containing 0.5 mm mgcl2 and 0.9 mm cacl2 , 0.5 mg / ml bovine serum albumin , and 
 1 mg / ml glucose each time to remove any growth factors which may interfere 
 with subsequent assays .
following the final wash , the supernatant 
 was removed ; cells resuspended in 10 ml of assay buffer and counted 
 using a hemocytometer .
cells were diluted to give a final cell number 
 in the assay of 1  10 cells per tube . a positive 
 control consisted of 100 nm unlabeled neurotrophin , negative control 
 was assay buffer , added to the cells before 2 nm i - labeled 
 neurotrophin was pipetted into each tube .
three 100 l aliquots from each 
 tube were spun through 5% sucrose in assay buffer to pellet the cells 
 ( 20000 g at 4 c ) , frozen in solid co2ethanol bath , and snipped ( using adapted clippers to maintain 
 reproducible depth of tip ) .
i - ngf in the cell pellet 
 was counted with a gamma counter ( lkb wallac 1272 ) .
the assay was 
 deemed to have been successful if the unlabeled ngf in the positive 
 control was able to displace > 90% of the counts compared with the 
 negative control .
hek293 cells were resuspended in complete 
 dmem containing 10% fcs , 2 mm glutamine , 10 u / ml penicillin , 0.1 mg / ml 
 streptomycin , and 0.5 mg / ml g418 as necessary .
cells were plated out 
 at 1  10 in 100 l per well into polylysine 
 coated black - walled / clear bottomed 96-well plates ( corning cc692 ) 
 and incubated at 37 c overnight in 5% co2 .
once cells 
 had reached 7080% confluence the serum concentration was reduced 
 to 0.1% fcs overnight .
half the plate was populated with parent 
 cells and the other half with cells expressing full length glycosylated 
 human trka .
parent and trka cells were exposed to the same controls 
 and compounds to ensure the results expressed were receptor - specific 
 and not due to , e.g. , toxicity .
responses 
 in triplicate in the presence and absence of neurotrophin to detect 
 antagonist and agonist activity under the same conditions .
amitriptyline 
 was added to the cells in pbs in 50 l , with 
 or without 0.5 nm ngf , respectively , for assessing antagonist or agonist 
 activity and incubated for 5 min at 37 c .
cells were washed 
 once in ice - cold pbs ( 200 l ) and then placed on ice .
cells 
 were fixed using 50 l per well of 4% ( w / v ) paraformaldehyde 
 in pbs and incubated for 30 min at room temperature .
cells were washed 
 once with pbs ( 100 l ) and permeabilized by adding 50 l 
 per well 20 c methanol for 5 min followed by a further 
 wash in pbs ( 100 l ) .
nonspecific binding was reduced by incubating 
 with 50 l per well 10% goat serum in pbs at room temperature 
 ( 2 h ) and washed once with pbs ( 100 l ) before 30 l per 
 well at 1:500 dilution anti - phosphoerk ( perk ) antibody ( new england 
 biolabs ltd . )
the cells were washed three 
 times in pbs prior to application of 30 l per well of the secondary 
 antibody goat anti - rabbit alexa 546 ( invitrogen ) at 1/300 in pbs plus 
 1% goat serum and 4,6-diamidino-2-phenylindole ( dapi ) ( sigma - aldrich ) 
 1 mg / ml diluted to 1/10000 for nuclear staining .
plates were incubated 
 for 90 min in the dark at room temperature and then washed three times 
 with 200 l of pbs per well and stored in 200 l of pbs 
 at 4 c until read in the incell 1000 analyzer ( ge healthcare ) .
data were collected using excitation  535 nm and emission 
  620 nm for 2000 ms exposure for alexafluor secondary antibody 
 signal for erk activation ( perk 1/2 ) .
dapi ( as nuclear stain ) was 
 assessed using  excitation 360 nm and emission  460 
 nm with 600 ms exposure .
data were analyzed and presented as perk 
 intensity per cell . for 
 effects of trkaig2-nmr on ngf - dependent pc12 cell proliferation or 
 survival , celltiter
96 aqueous nonradioactive cell proliferation reagents 
 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium , inner salt ( mts ) and the electron coupling 
 reagent phenazinemethosulfate ( pms ) ( sigma ) were added to cells as 
 per manufacturer s instructions .
cells were seeded at a cell 
 density of 10000 cells per well into 96-well plates containing 100 
 l of complete cell culture media ( dmem , 1% ( v : v ) penicillin / streptomycin 
 glutamine ) .
mouse ngf was added to give a final concentration of 5 
 ng / ml .
mts and pms solutions were then added with final concentrations 
 of 333 g / ml mts and 25 m pms .
the assay was performed 
 in triplicate at incubation times of 1 , 2 , and 3 h at 37 c with 
 5% co2 to allow color development .
absorbance at 490 nm 
 was recorded on a biohit bp-800 spectrophotometer ( helsinki , finland ) .
n labeled trkaig2-nmr 
 construct was diluted to achieve a final concentration in the assay 
 of 4.5 m , 2.25 m , 1.125 m , 562 nm , 281 nm , and 
 140 nm .
ngf ( human recombinant , sigma ) evokes neurite outgrowth on 
 pc12 cells at a concentration of 0.04 nm after 3 days incubation at 
 37 c .
buffer controls with and without ngf were used to verify 
 that any suppression of neurite outgrowth was due to the protein and 
 not the buffer in which it was dissolved .
other control wells consisting of pc12 cells with and 
 without ngf ( in media ) were included to check for cell responsiveness . 

the cells were removed from 90% confluent t75 flask in versene and 
 resuspended in complete media ( dulbecco s modified eagle medium 
 ( dmem ) ( gibco brl ) supplemented with 10% ( v / v ) fetal bovine serum 
 ( fbs , sigma ) , 1% ( v / v ) penicillin / streptomycin solution ( sigma ) , and 
 2 mm l - glutamine ( gibco brl ) .
the cell number was adjusted 
 to 4  10 per ml and 0.5 ml cells were seeded into 
 collagen coated 24-well plates , 0.5 ml per well .
neurite outgrowth was assessed semiquantitatively on a scale of 04 
 as described previously the assay was 
 valued in quadruplicate with wells blinded by two independent assessors .
 the tyrosine kinase a ( trka ) receptor 
 is a validated therapeutic 
 intervention point for a wide range of conditions . 
 trka activation 
 by nerve growth factor ( ngf ) binding the second extracellular immunoglobulin 
 ( trkaig2 ) domain triggers intracellular signaling cascades . in the 
 periphery , this promotes the pain phenotype and , in the brain , cell 
 survival or differentiation . 
 reproducible structural information and 
 detailed validation of protein  ligand interactions aid drug 
 discovery . 
 however , the isolated trkaig2 domain crystallizes as a -strand - swapped 
 dimer in the absence of ngf , occluding the binding surface . here 
 we 
 report the design and structural validation by nuclear magnetic resonance 
 spectroscopy of the first stable , biologically active construct of 
 the trkaig2 domain for binding site confirmation . 
 our structure closely 
 mimics the wild - type fold of trkaig2 in complex with ngf ( 1www.pdb ) , and the 1h15n correlation spectra confirm that both 
 ngf and a competing small molecule interact at the known binding interface 
 in solution . 
Introduction
Results
and Discussion
Conclusions
Materials and Methods