title: 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.
authors.affiliation: School of Clinical Sciences, Level 2, Learning and Research, Southmead Hospital , Bristol BS10 5NB, United Kingdom.
authors.forename: Debbie K
authors.initials: DK
authors.lastname: Shoemark
authors.forename: Christopher
authors.initials: C
authors.lastname: Williams
authors.forename: Mark S
authors.initials: MS
authors.lastname: Fahey
authors.forename: Judy J
authors.initials: JJ
authors.lastname: Watson
authors.forename: Sue J
authors.initials: SJ
authors.lastname: Tyler
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Scoltock
authors.forename: Rosamund Z
authors.initials: RZ
authors.lastname: Ellis
authors.forename: Elaine
authors.initials: E
authors.lastname: Wickenden
authors.forename: Antony J
authors.initials: AJ
authors.lastname: Burton
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Hemmings
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Bailey
authors.forename: David
authors.initials: D
authors.lastname: Dawbarn
authors.forename: David E
authors.initials: DE
authors.lastname: Jane
authors.forename: Christine L
authors.initials: CL
authors.lastname: Willis
authors.forename: Richard B
authors.initials: RB
authors.lastname: Sessions
authors.forename: Shelley J
authors.initials: SJ
authors.lastname: Allen
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Crump
mesh_terms: D000639:Amitriptyline / Q000378:metabolism; D001665:Binding Sites; D015195:Drug Design; D055808:Drug Discovery; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D020932:Nerve Growth Factor / Q000378:metabolism; D017434:Protein Structure, Tertiary; D020917:Receptor, trkA / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; C498075:tyrosine receptor kinase A Ig2; D000639:Amitriptyline; D020932:Nerve Growth Factor; D020917:Receptor, trkA