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

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.

Figure 1

(A) Sequence comparison of “wild-type” 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). (B) ¹H–¹⁵N HSQC spectra of the wild-type construct TrkAIg2-WT. The amide peak dispersal indicates that the protein is not a homogeneous population or that some of the structure is disordered.

Figure 2

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 fold is predicted to be minimally perturbed when this disulfide is introduced. 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 β-strand in the NGF bound crystal structure (1WWW.pdb).

Figure 3

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 μM) and the TrkAIg2-NMR (open circles, EC₅₀ 2.1 μM) 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 ¹⁵N labeled TrkAIg2. The TrkAIg2-NMR construct inhibited neurite outgrowth with an EC₅₀ 1.59 μM. Inset: ¹⁵N-labeled TrkAIg2-NMR mediated sequestration of ¹²⁵I-labeled NGF in a competition assay against full-length human TrkA receptors expressed on HEK cells, with an approximate IC₅₀ of 2 μM. (C) Complex formation between TrkAIg2-NMR and NGF. 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. (D) ¹H–¹⁵N HSQC spectra of TrkAIg2-NMR, showing vastly improved homogeneity of peak intensity and line shape.

Figure 4

Solution structure of TrkAIg2-NMR and comparison with the crystal structure. (A) The ensemble of 20 TrkAIg2-NMR domain structures. The NGF binding groove is indicated with a dotted line. (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.

Figure 5

Interaction of NGF with TrkAIg2-NMR. (A) the overlaid ¹H–¹⁵N 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) Δδ_NH 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 but no CSP. 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 ¹H–¹⁵N HSQC spectrum drawn as sticks.

Figure 6

TrkAIg2-NMR constructs interacts with a small molecule, amitriptyline. (A) Peak shifts attributed to amitriptyline binding. (B) Δδ_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 μM. (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 μM.