Towards structure determination of protein-ligand complexes of TrkA and TrkB receptors: from protein production to crystal optimization

Neurotrophins (NGF, BDNF, NT3 and NT4/5) are a family of closely related growth factors, that promote neuronal proliferation, differentiation, survival and apoptosis, as well as formation and modulation of synaptic networks in peripheral and central nervous system. Key mediators of these pleiotropic effects are at one side the tropomyosin kinase receptors (TrkA, TrkB and TrkC), that are selectively and specifically activated by neurotrophins via binding to the d5 sub-domain of the extracellular region, and on the other side p75 neurotrophin receptor, characterized by unspecific and lower affinity interactions that generally lead to death signals. Their pivotal role in development and maintenance of the nervous system has made NT-Trks axes pharmacological targets of great interest to achieve neuroprotective and neurogenic effects in pathological conditions. To address the current lack of therapeutic agents for treating and slowing down neurodegenerative diseases, the “EuroNeurotrophin” (ENT) network aimed to discover new small compounds with neurotrophin-like effects. Within the network, several mimetics with agonist effects on TrkA and TrkB receptors have been developed, but biophysical and structural information is missing. Therefore, key objective of the PhD project was to elucidate the structural features for ligand binding by X-ray crystallography and to integrate such structural information with biophysical characterization of binding affinity values and kinetic profiles, as well as to provide the first structure at high resolution of the complex between BDNF and TrkB extracellular domain. First, efforts focused on developing purification protocols for the production at high yields of the d5 domains of TrkA and TrkB in E. coli expression system. By varying intrinsic proprieties and extrinsic factors affecting the solubility of the recombinant proteins, different constructs of TrkA-d5 and TrkB-d5 were designed and several expression, purification and refolding strategies were tested. Among all, the co-expression approach resulted in increased solubility of the TrkB-d5 and crystallization screens were set up, while a low yield of natively fold TrkA-d5 monomer was obtained by protein recovery and refolding from inclusion bodies, no sufficient for structural studies. Second, the whole extracellular domains of TrkA and TrkB receptors were produced in Hek293 cells suspension, using a glycosidase inhibitor to decrease the complexity and heterogeneity of the N-glycosylation. The recombinant ectodomains were used for crystallization screening and preliminary studies by cryo-electron microscopy, as well as for biophysical characterization of the binding of mimetic compounds by Grating Coupled Interferometry. Showing binding affinity values in micromolar range, two ENT small compounds BNN27 and ENT-C117 were confirmed to be TrkA and TrkB selective ligands, respectively, and ENT-A010 compound to bind with similar affinity both receptors. As regards as the structural studies, both TrkA-NGF and TrkB-BDNF complexes consisted of small and heterogeneous particles, which could not allow a structure reconstruction by Cryo-EM. From the crystallization screening, no diffracting crystals of TrkA and TrkB were obtained. However, several precipitating conditions of TrkA-NGF complex gave rise to needle-like and plate crystals, which were used for microseeding matrix screening, opening up an iterative process that may lead to crystal formation.