High-affinity anti-Arc nanobodies provide tools for structural and functional studies

Area of Science:

• Neuroscience
• Structural Biology
• Molecular Biology

Background:

• Activity-regulated cytoskeleton-associated protein (Arc) is crucial for synaptic plasticity and memory.
• The precise mechanisms and structures underlying Arc function remain largely unknown.
• Arc's N-terminal domain (NTD) mediates membrane binding, while the C-terminal domain (CTD) interacts with postsynaptic ligands.

Purpose of the Study:

• To generate novel tools for investigating Arc structure and function.
• To elucidate the structural dynamics and oligomerization mechanisms of Arc.
• To explore the formation of Arc's retrovirus-like capsids involved in intercellular signaling.

Main Methods:

• Production and characterization of six high-affinity anti-Arc nanobodies (Nbs).
• Crystallization of rat and human Arc CTD in complex with Nbs.
• X-ray crystallography, Small-Angle X-ray Scattering (SAXS), and molecular dynamics simulations.

Main Results:

• Two anti-Arc Nbs were successfully used to crystallize Arc CTD.
• One Nb identified a binding site within the stargazin-binding pocket, suggesting competitive ligand interaction.
• Crystallization revealed two distinct conformations of human Arc CTD, including a capsid-like state.

Conclusions:

• Recombinant anti-Arc nanobodies are versatile tools for studying Arc structure, function, and capsid formation.
• Structural dynamics of the CTD and NTD dimerization appear to facilitate capsid assembly.
• These Nbs can be engineered as genetically encoded inhibitors for studying Arc's role in neuronal plasticity.