A sulfotyrosine motif in the human TrkB extracellular domain is required for activation by the agonist antibody ZEB85

The brain-derived neurotrophic factor (BDNF)-tropomyosin receptor kinase B (TrkB) signaling axis is a key effector of synaptic plasticity and neuroprotection. While TrkB activation is a major target to prevent neural dysfunction, stimulation with exogenous BDNF administration has not proven tractable given the unfavorable physiochemical properties of BDNF. In addition, BDNF also activates a cell death pathway by binding to the neurotrophin receptor p75. The TrkB agonist antibody ZEB85 provides an alternative route to the selective activation of TrkB. To understand the ZEB85 mechanism of action, we mapped the binding site for ZEB85 to the TrkB extracellular juxtamembrane domain and found a sulfotyrosine modification at residue 400 crucial for ZEB85 interaction and activation of TrkB signaling. We determined the structures of ZEB85 and BDNF bound to their respective binding sites on human TrkB suggesting independent sites for each ligand, despite prior evidence of direct competition. Using structure-guided BDNF- and ZEB85-binding deficient TrkB mutants, we assessed their ability to sequester ligands from full-length TrkB in cultured human neurons. We found that the BDNF binding site extends into the juxtamembrane domain of TrkB but does not require the sulfotyrosine 400 to activate TrkB. Together with biophysical analysis and AlphaFold modeling, these results explain how BDNF can displace ZEB85 from TrkB through an overlapping epitope. Our findings reveal unique features of TrkB, not present in the related neurotrophin receptors TrkA and TrkC and suggest future directions to explore the role of sulfotyrosine in TrkB signaling.