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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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Interactionof NGF with TrkAIg2-NMR. (A) the overlaid 1H–15N HSQC spectra of TrkAIg2-NMR construct before(black) and after (red) the addition of stoichiometrically equivalentquantity of NGF. Resonances that show chemical shift perturbations(CSPs) or appear significantly broadened have been annotated. (B)ΔδNH between free TrkAIg2-NMR and NGF present(orange), where the height is proportional to the difference in ppm.Negative green peaks indicate that line-broadening was observed butno CSP. The majority of the residues that show CSPs or line broadeninginteract with the N-terminus of NGF that forms a helix on bindingthe TrkAIg2 domain. (C) Surface representation of TrkAIg2-NMR (dark-gray)and NGF (cyan) with CSPs and line broadening shaded orange and exchange-broadenedpeaks only shaded green. (D) NGF (cyan) is depicted in secondary structureas a ribbon in complex with one TrkAIg2-NMR (green), with residuescorresponding to the peaks shifted in the 1H–15N HSQC spectrum drawn as sticks.
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fig5: Interactionof NGF with TrkAIg2-NMR. (A) the overlaid 1H–15N HSQC spectra of TrkAIg2-NMR construct before(black) and after (red) the addition of stoichiometrically equivalentquantity of NGF. Resonances that show chemical shift perturbations(CSPs) or appear significantly broadened have been annotated. (B)ΔδNH between free TrkAIg2-NMR and NGF present(orange), where the height is proportional to the difference in ppm.Negative green peaks indicate that line-broadening was observed butno CSP. The majority of the residues that show CSPs or line broadeninginteract with the N-terminus of NGF that forms a helix on bindingthe TrkAIg2 domain. (C) Surface representation of TrkAIg2-NMR (dark-gray)and NGF (cyan) with CSPs and line broadening shaded orange and exchange-broadenedpeaks only shaded green. (D) NGF (cyan) is depicted in secondary structureas a ribbon in complex with one TrkAIg2-NMR (green), with residuescorresponding to the peaks shifted in the 1H–15N HSQC spectrum drawn as sticks.

Mentions: We then tested whetherwe could detect a direct protein–protein interaction in solutionunder conditions used for NMR with the TrkA receptor’s cognateligand, the cytokine NGF (from mouse submaxillary salivary glands22). NGF readily adsorbs onto many surfaces,23 but reversible acid denaturation reduces thispropensity. NGF is therefore stored in sodium acetate at pH 2 to minimizelosses. A single-shot NMR assay was recorded to ensure that no pHperturbation occurred when the complex was formed as 650 μgof NGF (preadjusted to pH 6.9) was added to a solution of 15N-TrkAIg2-NMR to approach a 1:1 stoichiometry. Complex formationwas confirmed by gel filtration (Figure 3C).Figure 5A shows the 1H–15N HSQC spectra before (black) and after (red) the additionof a stoichiometric concentration of NGF. A number of residues showeither amide chemical shift perturbations (CSPs) or are significantlyline broadened in the presence of NGF (Figure 5B). These perturbed residues were mapped onto the crystal structureof the TrkAIg2 domain bound to NGF and are shown in Figure 5C,D. Although the CSPs are <0.15 ppm,24 the predominant spectral changes cluster aroundthe groove formed by strands β2, β4, and β5 thatbinds the N-terminal helix of NGF with several additional residuesin the loop connecting strands β5 and β6.5 In particular, residues which are almost completely linebroadened (shown as green bars in Figure 5B)form a patch centered in the binding groove which would contact thehelix (Figure 5C). This N-terminal helix isunstructured when NGF is crystallized in the free form (1BET.pdb), and electrondensity is only visible for this region when NGF is in complex withthe TrkAIg2 domain (1WWW.pdb) and makes an energetically important contribution to the bindingenergy of the TrkAIg2/NGF interaction.25 We note however that the observed peak broadening is not as pronouncedas expected if a large, stable complex was formed with NGF (Kd low micromolar), suggesting that not all ofthe NGF was available in solution, perhaps due to binding to the glassof the NMR tube and the TrkAIg2-NMR remains well in excess.


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)

Interactionof NGF with TrkAIg2-NMR. (A) the overlaid 1H–15N HSQC spectra of TrkAIg2-NMR construct before(black) and after (red) the addition of stoichiometrically equivalentquantity of NGF. Resonances that show chemical shift perturbations(CSPs) or appear significantly broadened have been annotated. (B)ΔδNH between free TrkAIg2-NMR and NGF present(orange), where the height is proportional to the difference in ppm.Negative green peaks indicate that line-broadening was observed butno CSP. The majority of the residues that show CSPs or line broadeninginteract with the N-terminus of NGF that forms a helix on bindingthe TrkAIg2 domain. (C) Surface representation of TrkAIg2-NMR (dark-gray)and NGF (cyan) with CSPs and line broadening shaded orange and exchange-broadenedpeaks only shaded green. (D) NGF (cyan) is depicted in secondary structureas a ribbon in complex with one TrkAIg2-NMR (green), with residuescorresponding to the peaks shifted in the 1H–15N HSQC spectrum drawn as sticks.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4504729&req=5

fig5: Interactionof NGF with TrkAIg2-NMR. (A) the overlaid 1H–15N HSQC spectra of TrkAIg2-NMR construct before(black) and after (red) the addition of stoichiometrically equivalentquantity of NGF. Resonances that show chemical shift perturbations(CSPs) or appear significantly broadened have been annotated. (B)ΔδNH between free TrkAIg2-NMR and NGF present(orange), where the height is proportional to the difference in ppm.Negative green peaks indicate that line-broadening was observed butno CSP. The majority of the residues that show CSPs or line broadeninginteract with the N-terminus of NGF that forms a helix on bindingthe TrkAIg2 domain. (C) Surface representation of TrkAIg2-NMR (dark-gray)and NGF (cyan) with CSPs and line broadening shaded orange and exchange-broadenedpeaks only shaded green. (D) NGF (cyan) is depicted in secondary structureas a ribbon in complex with one TrkAIg2-NMR (green), with residuescorresponding to the peaks shifted in the 1H–15N HSQC spectrum drawn as sticks.
Mentions: We then tested whetherwe could detect a direct protein–protein interaction in solutionunder conditions used for NMR with the TrkA receptor’s cognateligand, the cytokine NGF (from mouse submaxillary salivary glands22). NGF readily adsorbs onto many surfaces,23 but reversible acid denaturation reduces thispropensity. NGF is therefore stored in sodium acetate at pH 2 to minimizelosses. A single-shot NMR assay was recorded to ensure that no pHperturbation occurred when the complex was formed as 650 μgof NGF (preadjusted to pH 6.9) was added to a solution of 15N-TrkAIg2-NMR to approach a 1:1 stoichiometry. Complex formationwas confirmed by gel filtration (Figure 3C).Figure 5A shows the 1H–15N HSQC spectra before (black) and after (red) the additionof a stoichiometric concentration of NGF. A number of residues showeither amide chemical shift perturbations (CSPs) or are significantlyline broadened in the presence of NGF (Figure 5B). These perturbed residues were mapped onto the crystal structureof the TrkAIg2 domain bound to NGF and are shown in Figure 5C,D. Although the CSPs are <0.15 ppm,24 the predominant spectral changes cluster aroundthe groove formed by strands β2, β4, and β5 thatbinds the N-terminal helix of NGF with several additional residuesin the loop connecting strands β5 and β6.5 In particular, residues which are almost completely linebroadened (shown as green bars in Figure 5B)form a patch centered in the binding groove which would contact thehelix (Figure 5C). This N-terminal helix isunstructured when NGF is crystallized in the free form (1BET.pdb), and electrondensity is only visible for this region when NGF is in complex withthe TrkAIg2 domain (1WWW.pdb) and makes an energetically important contribution to the bindingenergy of the TrkAIg2/NGF interaction.25 We note however that the observed peak broadening is not as pronouncedas expected if a large, stable complex was formed with NGF (Kd low micromolar), suggesting that not all ofthe NGF was available in solution, perhaps due to binding to the glassof the NMR tube and the TrkAIg2-NMR remains well in excess.

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