Structure of monomeric full-length ARC sheds light on molecular flexibility, protein interactions, and functional modalities

Abstract

The activity-regulated cytoskeleton-associated protein (ARC) is critical for long-term synaptic plasticity and memory formation. Acting as a protein interaction hub, ARC regulates diverse signalling events in postsynaptic neurons. A protein interaction site is present in the ARC C-terminal domain (CTD), a bilobar structure homologous to the retroviral Gag capsid domain. However, knowledge of the 3-dimensional structure of full-length ARC is required to elucidate its molecular function. We purified recombinant monomeric full-length ARC and analyzed its structure using small-angle X-ray scattering and synchrotron radiation circular dichroism spectroscopy. In solution, monomeric ARC has a compact, closed structure, in which the oppositely charged N-terminal domain (NTD) and CTD are juxtaposed, and the flexible linker between them is not extended. The modelled structure of ARC is supported by intramolecular live-cell FRET imaging in rat hippocampal slices. Peptides from several postsynaptic proteins, including stargazin, bind to the N-lobe, but not to the C-lobe, of the bilobar CTD. This interaction does not induce large-scale conformational changes in the CTD or flanking unfolded regions. The ARC NTD contains long helices, predicted to form an anti-parallel coiled coil; binding of ARC to phospholipid membranes requires the NTD. Our data support a role for the ARC NTD in oligomerization as well as lipid membrane binding. These findings have important implications for the structural organization of ARC in distinct functional modalities, such as postsynaptic signal transduction and virus-like capsid formation.