The AP-4 accessory protein tepsin exhibits multivalent binding to LC3B.

Tepsin is an accessory protein in Adaptor Protein 4 (AP-4) coated vesicles responsible for trafficking cargo from the trans-Golgi network (TGN). AP-4 vesicles recognize and sort multiple cargoes including ATG9A, a lipid scramblase essential for autophagosome maturation. In cultured cells, tepsin loss alters ATG9A distribution and autophagosome morphology, and tepsin has been shown to contain a canonical LC3-interacting region (LIR) motif required for proper ATG9A distribution. Computational modeling in AlphaFold Multimer combined with biochemical and biophysical experiments identified three additional LC3B binding motifs within tepsin disordered regions. Structural models paired with bio-layer interferometry (BLI) uncovered and confirmed specific residues involved in each interaction and indicated all four motifs independently engage the LC3B LIR docking site (LDS). Thermodynamic and kinetic properties associated with each motif found in full-length tepsin were quantified. BLI and biochemical data reveal all four motifs in tepsin must be mutated to abrogate binding to LC3B in vitro, while stoichiometry data estimate one tepsin likely binds two LC3B at one time on a surface or membrane. Together, data suggest tepsin could respond dynamically to LC3B concentrations on membranes by leveraging multivalency to modulate binding strength.