Distinct competitive and allosteric binding modes of nanobodies targeting EphA4

EphA4 signaling is a key negative regulator of axonal regeneration and a genetic modifier of amyotrophic lateral sclerosis (ALS), making EphA4 an attractive but mechanistically underexplored therapeutic target. Although EphA4-targeting nanobodies have shown inhibitory potential, the structural principles governing their binding and inhibitory mechanisms remain largely unknown. Here, we report high-resolution crystal structures of the EphA4 ligand-binding domain (LBD) in complex with four nanobodies (Nb50, Nb53, Nb57, and Nb60), resolved at 1.34-2.21 Å. Nb50, Nb53, and Nb57 competitively engage the canonical ephrin-binding pocket through deep CDR3 insertion, directly mimicking ephrin recognition. In contrast, Nb60 adopts a previously unrecognized binding mode, contacting two EphA4 molecules at noncanonical sites in the crystal structure through framework-dominated interactions and supporting a structural model for steric and allosteric restriction of ephrin access. Integrated biophysical analyses reveal distinct thermodynamic and kinetic signatures underlying these binding mechanisms. Together, our findings uncover unexpected structural diversity in nanobody-mediated EphA4 recognition and provide a framework for rational development of EphA4-targeted modulators for ALS and related neurodegenerative conditions.