RESUMEN
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 ß-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.
Asunto(s)
Descubrimiento de Drogas , Espectroscopía de Resonancia Magnética/métodos , Receptor trkA/química , Amitriptilina/metabolismo , Sitios de Unión , Diseño de Fármacos , Factor de Crecimiento Nervioso/metabolismo , Estructura Terciaria de Proteína , Receptor trkA/metabolismo , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
Monoclonal antibodies are reproducible, specific, and cost-effective molecular probes; use outside the laboratory is, however, restricted by technical limitations. Addressing these constraints, the first self-signalling antibodies are now described, where specific antigen binding causes release of bound reporter from bispecific antibodies (BsAb) to generate a detectable signal. The report examines the concept that two different antibody binding sites in close proximity can promote interaction between molecules recognised by these sites, generating a signal by molecular crowding. Signal strength is found to increase with increasing homogeneity for a BsAb reactive with multimeric surfactant antigen; signal response is linear for a BsAb reactive with univalent small analyte deoxypyridinoline. Self-signalling is consistent with intramolecular steric hindrance. This is the first report detailing integration of two different functions, molecular detection and signal response, into BsAbs and with detection of large and small analytes, has generic application to antibody-based systems.