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

Targeting the Nerve Growth Factor (NGF) Pathway in Drug Discovery. Potential Applications to New Therapies for Chronic Pain

“…More recently, Shoemark and co-workers have used nuclear magnetic resonance (NMR) to inform a solution structure of a 13 C, 15 N-labeled TrkAIg2 analogue, known as TrkAIg2-NMR . This construct demonstrated wild-type TrkAIg2 activity such as NGF binding and inhibition of NGF-induced neurite outgrowth.…”

“…42,68 More recently, Shoemark and co-workers have used nuclear magnetic resonance (NMR) to inform a solution structure of a 13 C, 15 N-labeled TrkAIg2 analogue, known as TrkAIg2-NMR. 69 This construct demonstrated wild-type TrkAIg2 activity such as NGF binding and inhibition of NGF-induced neurite outgrowth. 1 H− 15 N heteronuclear single quantum correlation (HSQC) spectra showed significant chemical shift perturbations (CSPs) or line broadening in the presence of NGF, with most of the spectral changes occurring in the region that binds the N-terminal helix of NGF.…”

3β, 6β-dichloro-5-hydroxy-5α-cholestane facilitates neuronal development through modulating TrkA signaling regulated proteins in primary hippocampal neuron

“…Several earlier reports highlighted the critical contributions of the fifth subdomain (extracellular part) at the extracellular part of TrkA to NGF binding in initiating NGF mediated TrkA activation 33–36 . Furthermore, previous NMR studies also revealed that the fifth subdomain contains small molecule binding site 37 , where agonist can bind and modulate TrkA activity. Thence, molecular modeling studies were further incorporated to get insight into the interaction patterns and efficiency of SCH to the TrkA’s fifth subdomain.…”

Deciphering Molecular Mechanism of the Neuropharmacological Action of Fucosterol through Integrated System Pharmacology and In Silico Analysis

“…Several earlier reports highlighted that the fifth subdomain at the extracellular part of the TrkB receptor plays major role in the BDNF mediated TrkB interactions [39,40,41,42]. This domain furnishes a small molecule binding site [43]. The binding site is hydrophobic, where the residues Asn350, Pro351, Thr352, and Met354 directly involved in contact with BDNF [39].…”