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Design and nuclear magnetic resonance (NMR) structure determination of the second extracellular immunoglobulin tyrosine kinase A (TrkAIg2) domain construct for binding site elucidation in drug discovery.

Shoemark DK, Williams C, Fahey MS, Watson JJ, Tyler SJ, Scoltock SJ, Ellis RZ, Wickenden E, Burton AJ, Hemmings JL, Bailey CD, Dawbarn D, Jane DE, Willis CL, Sessions RB, Allen SJ, Crump MP - J. Med. Chem. (2014)

Bottom Line: 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.Our structure closely mimics the wild-type fold of TrkAIg2 in complex with NGF ( 1WWW .pdb), and the (1)H-(15)N correlation spectra confirm that both NGF and a competing small molecule interact at the known binding interface in solution.

View Article: PubMed Central - PubMed

Affiliation: School of Clinical Sciences, Level 2, Learning and Research, Southmead Hospital , Bristol BS10 5NB, United Kingdom.

ABSTRACT
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 β-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 (1)H-(15)N correlation spectra confirm that both NGF and a competing small molecule interact at the known binding interface in solution.

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Comparison of X-ray crystallographicstructures and modeled TrkA-Ig2in free forms and complexed with NGF. (A) Crystal structure of thecomplex between NGF and TrkA-Ig2 (1WWW.pdb)20 withtwo molecules of TrkA-Ig2 shown in magenta in binding to an NGF dimer(slate). (B) Crystal structure of two molecules of TrkA-Ig2 (red andlight-orange) in the absence of NGF forming a strand-swapped dimer(1WWA.pdb).4 (C) Superposition of the X-ray structure of afurther strand-swapped dimer of TrkAIg2 (PDB code 1HE7)20 (cyan) with the position of P285 and F367 highlighted.The structure of modeled domain 5 (green) is shown incorporating theP285C and F367C mutations and the new disulfide bond after energyminimization. The fold is predicted to be minimally perturbed whenthis disulfide is introduced. The N-terminal strand in the modeledconstruct is highlighted with *, and the point at which the non- andstrand-swapped dimers diverge is indicated by **. (D) Similarly, superpositionof the minimized disulfide bridged construct shows close structuralsimilarity with the packing of the N-terminal β-strand in theNGF bound crystal structure (1WWW.pdb).
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fig2: Comparison of X-ray crystallographicstructures and modeled TrkA-Ig2in free forms and complexed with NGF. (A) Crystal structure of thecomplex between NGF and TrkA-Ig2 (1WWW.pdb)20 withtwo molecules of TrkA-Ig2 shown in magenta in binding to an NGF dimer(slate). (B) Crystal structure of two molecules of TrkA-Ig2 (red andlight-orange) in the absence of NGF forming a strand-swapped dimer(1WWA.pdb).4 (C) Superposition of the X-ray structure of afurther strand-swapped dimer of TrkAIg2 (PDB code 1HE7)20 (cyan) with the position of P285 and F367 highlighted.The structure of modeled domain 5 (green) is shown incorporating theP285C and F367C mutations and the new disulfide bond after energyminimization. The fold is predicted to be minimally perturbed whenthis disulfide is introduced. The N-terminal strand in the modeledconstruct is highlighted with *, and the point at which the non- andstrand-swapped dimers diverge is indicated by **. (D) Similarly, superpositionof the minimized disulfide bridged construct shows close structuralsimilarity with the packing of the N-terminal β-strand in theNGF bound crystal structure (1WWW.pdb).

Mentions: To engineer a construct for use in the absenceof NGF, we re-examinedknown crystal structures of these proteins. The X-ray crystallographicstudy of the wild-type single TrkA-Ig2 domain (Figure 2A) bound to NGF previously revealed two molecules of the TrkA-Ig2domain bound to a central NGF dimer.5 Anextensive interface was observed between NGF and the individual TrkA-Ig2domains, but there were no direct contacts between the TrkA-Ig2 domainsthemselves. When studied in isolation, however, the Ig2 domains fromTrkA, TrkB, and TrkC have all been shown to form strand-swapped dimersin solved crystal structures (Figure 2B), anassociation that occludes the interaction site on TrkA for NGF. Therefore,at high concentrations, the formation of oligomeric4 species in addition to unstructured regions might explainthe poor NMR characteristics of the TrkAIg2-WT construct. Figure 2C shows an overlay of a single TrkA-Ig2 domain chainfrom the strand-swapped crystal structure 1HE7.pdb with a single chain from the crystalstructure 1WWW.pdb in complex with NGF. The strand-swapped structure of TrkA-Ig2(residues 285–413), studied by Robertson et al., revealed astable core structure extending to P382 after which no electron densitywas observed.20 The C-terminal was presumedto be flexible and might not therefore be ideal for solution-stateNMR studies. Therefore, the C-terminal was truncated to end in theresidues DNPF (383) (Figure 1A). To preventβ-strands swapping between adjacent monomers at high concentrations,an additional disulfide bond was also introduced to act as an intramolecularstaple between β-strand 1 (P285C) and the β-hairpin betweenstrands 6 and 7 (F367C, Figure 1A) to yieldour first-generation construct TrkAIg2-DS1. The disulfide bridge wasmodeled onto chain X of TrkA from the crystal structure 1WWW.pdb and then energyminimized using Discover 2.98 (Accelrys) (Figure 2D). These residues were chosen because they were suitablydistant from the NGF binding face of the protein and therefore lesslikely to influence the binding of compounds to the target site. P285and F367 were already almost the optimal distance apart required fordisulfide bond formation. In the crystal structure, their side chainswere oriented toward each other so that disulfide formation betweentwo cysteines at these positions would be predicted to cause minimaldistortion to the overall fold. Importantly, these residues fulfilledanother necessary criterion21 predictedto produce a hyper-stable native state, namely they were not involvedin the hydrogen bonding pattern of the β-sheet.


Design and nuclear magnetic resonance (NMR) structure determination of the second extracellular immunoglobulin tyrosine kinase A (TrkAIg2) domain construct for binding site elucidation in drug discovery.

Shoemark DK, Williams C, Fahey MS, Watson JJ, Tyler SJ, Scoltock SJ, Ellis RZ, Wickenden E, Burton AJ, Hemmings JL, Bailey CD, Dawbarn D, Jane DE, Willis CL, Sessions RB, Allen SJ, Crump MP - J. Med. Chem. (2014)

Comparison of X-ray crystallographicstructures and modeled TrkA-Ig2in free forms and complexed with NGF. (A) Crystal structure of thecomplex between NGF and TrkA-Ig2 (1WWW.pdb)20 withtwo molecules of TrkA-Ig2 shown in magenta in binding to an NGF dimer(slate). (B) Crystal structure of two molecules of TrkA-Ig2 (red andlight-orange) in the absence of NGF forming a strand-swapped dimer(1WWA.pdb).4 (C) Superposition of the X-ray structure of afurther strand-swapped dimer of TrkAIg2 (PDB code 1HE7)20 (cyan) with the position of P285 and F367 highlighted.The structure of modeled domain 5 (green) is shown incorporating theP285C and F367C mutations and the new disulfide bond after energyminimization. The fold is predicted to be minimally perturbed whenthis disulfide is introduced. The N-terminal strand in the modeledconstruct is highlighted with *, and the point at which the non- andstrand-swapped dimers diverge is indicated by **. (D) Similarly, superpositionof the minimized disulfide bridged construct shows close structuralsimilarity with the packing of the N-terminal β-strand in theNGF bound crystal structure (1WWW.pdb).
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Related In: Results  -  Collection

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fig2: Comparison of X-ray crystallographicstructures and modeled TrkA-Ig2in free forms and complexed with NGF. (A) Crystal structure of thecomplex between NGF and TrkA-Ig2 (1WWW.pdb)20 withtwo molecules of TrkA-Ig2 shown in magenta in binding to an NGF dimer(slate). (B) Crystal structure of two molecules of TrkA-Ig2 (red andlight-orange) in the absence of NGF forming a strand-swapped dimer(1WWA.pdb).4 (C) Superposition of the X-ray structure of afurther strand-swapped dimer of TrkAIg2 (PDB code 1HE7)20 (cyan) with the position of P285 and F367 highlighted.The structure of modeled domain 5 (green) is shown incorporating theP285C and F367C mutations and the new disulfide bond after energyminimization. The fold is predicted to be minimally perturbed whenthis disulfide is introduced. The N-terminal strand in the modeledconstruct is highlighted with *, and the point at which the non- andstrand-swapped dimers diverge is indicated by **. (D) Similarly, superpositionof the minimized disulfide bridged construct shows close structuralsimilarity with the packing of the N-terminal β-strand in theNGF bound crystal structure (1WWW.pdb).
Mentions: To engineer a construct for use in the absenceof NGF, we re-examinedknown crystal structures of these proteins. The X-ray crystallographicstudy of the wild-type single TrkA-Ig2 domain (Figure 2A) bound to NGF previously revealed two molecules of the TrkA-Ig2domain bound to a central NGF dimer.5 Anextensive interface was observed between NGF and the individual TrkA-Ig2domains, but there were no direct contacts between the TrkA-Ig2 domainsthemselves. When studied in isolation, however, the Ig2 domains fromTrkA, TrkB, and TrkC have all been shown to form strand-swapped dimersin solved crystal structures (Figure 2B), anassociation that occludes the interaction site on TrkA for NGF. Therefore,at high concentrations, the formation of oligomeric4 species in addition to unstructured regions might explainthe poor NMR characteristics of the TrkAIg2-WT construct. Figure 2C shows an overlay of a single TrkA-Ig2 domain chainfrom the strand-swapped crystal structure 1HE7.pdb with a single chain from the crystalstructure 1WWW.pdb in complex with NGF. The strand-swapped structure of TrkA-Ig2(residues 285–413), studied by Robertson et al., revealed astable core structure extending to P382 after which no electron densitywas observed.20 The C-terminal was presumedto be flexible and might not therefore be ideal for solution-stateNMR studies. Therefore, the C-terminal was truncated to end in theresidues DNPF (383) (Figure 1A). To preventβ-strands swapping between adjacent monomers at high concentrations,an additional disulfide bond was also introduced to act as an intramolecularstaple between β-strand 1 (P285C) and the β-hairpin betweenstrands 6 and 7 (F367C, Figure 1A) to yieldour first-generation construct TrkAIg2-DS1. The disulfide bridge wasmodeled onto chain X of TrkA from the crystal structure 1WWW.pdb and then energyminimized using Discover 2.98 (Accelrys) (Figure 2D). These residues were chosen because they were suitablydistant from the NGF binding face of the protein and therefore lesslikely to influence the binding of compounds to the target site. P285and F367 were already almost the optimal distance apart required fordisulfide bond formation. In the crystal structure, their side chainswere oriented toward each other so that disulfide formation betweentwo cysteines at these positions would be predicted to cause minimaldistortion to the overall fold. Importantly, these residues fulfilledanother necessary criterion21 predictedto produce a hyper-stable native state, namely they were not involvedin the hydrogen bonding pattern of the β-sheet.

Bottom Line: 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.Our structure closely mimics the wild-type fold of TrkAIg2 in complex with NGF ( 1WWW .pdb), and the (1)H-(15)N correlation spectra confirm that both NGF and a competing small molecule interact at the known binding interface in solution.

View Article: PubMed Central - PubMed

Affiliation: School of Clinical Sciences, Level 2, Learning and Research, Southmead Hospital , Bristol BS10 5NB, United Kingdom.

ABSTRACT
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 β-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 (1)H-(15)N correlation spectra confirm that both NGF and a competing small molecule interact at the known binding interface in solution.

Show MeSH
Related in: MedlinePlus