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

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Tepsin is an accessory protein in Adaptor Protein 4 (AP-4) coated vesicles, crucial for cargo trafficking from the trans-Golgi network (TGN).
• AP-4 vesicles handle cargoes like ATG9A, a lipid scramblase vital for autophagosome maturation.
• Tepsin's known LC3-interacting region (LIR) motif influences ATG9A distribution and autophagosome morphology.

Purpose of the Study:

• To investigate the molecular interactions between tepsin and LC3B.
• To characterize the binding kinetics and thermodynamics of tepsin-LC3B interactions.
• To understand how tepsin's multivalency contributes to its function in vesicle trafficking and autophagy.

Main Methods:

• Computational modeling using AlphaFold Multimer.
• Biochemical and biophysical experiments, including bio-layer interferometry (BLI).
• Quantification of thermodynamic and kinetic properties of tepsin-LC3B binding.

Main Results:

• Three additional LC3B binding motifs were identified in tepsin's disordered regions, in addition to the canonical LIR.
• All four motifs independently bind to the LC3B LIR docking site (LDS).
• Mutating all four motifs was necessary to abolish LC3B binding in vitro; tepsin likely binds two LC3B molecules simultaneously.

Conclusions:

• Tepsin utilizes multivalency through four distinct LC3B binding motifs to modulate binding strength.
• This multivalency enables tepsin to respond dynamically to cellular LC3B concentrations.
• These findings provide insights into the regulation of AP-4 vesicle trafficking and autophagosome maturation.