Arc self-association and formation of virus-like capsids are mediated by an N-terminal helical coil motif.

Activity-regulated cytoskeleton-associated protein (Arc) is a protein interaction hub with diverse roles in intracellular neuronal signaling, and important functions in neuronal synaptic plasticity, memory, and postnatal cortical development. Arc has homology to retroviral Gag protein and is capable of self-assembly into virus-like capsids implicated in the intercellular transfer of RNA. However, the molecular basis of Arc self-association and capsid formation is largely unknown. Here, we identified a 28-amino-acid stretch in the mammalian Arc N-terminal (NT) domain that is necessary and sufficient for self-association. Within this region, we identified a 7-residue oligomerization motif, critical for the formation of virus-like capsids. Purified wild-type Arc formed capsids as shown by transmission and cryo-electron microscopy, whereas mutant Arc with disruption of the oligomerization motif formed homogenous dimers. An atomic-resolution crystal structure of the oligomerization region peptide demonstrated an antiparallel coiled-coil interface, strongly supporting NT-NT domain interactions in Arc oligomerization. The NT coil-coil interaction was also validated in live neurons using fluorescence lifetime FRET imaging, and mutation of the oligomerization motif disrupted Arc-facilitated endocytosis. Furthermore, using single-molecule photobleaching, we show that Arc mRNA greatly enhances higher-order oligomerization in a manner dependent on the oligomerization motif. In conclusion, a helical coil in the Arc NT domain supports self-association above the dimer stage, mRNA-induced oligomerization, and formation of virus-like capsids. DATABASE: The coordinates and structure factors for crystallographic analysis of the oligomerization region were deposited at the Protein Data Bank with the entry code 6YTU.

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

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. We hypothesized that detailed knowledge of the three-dimensional molecular structure of monomeric full-length ARC is crucial to understand its function; therefore, we set out to determine the structure of ARC to understand its various functional modalities. We purified recombinant ARC and analyzed its structure using small-angle X-ray scattering and synchrotron radiation circular dichroism spectroscopy. Monomeric full-length 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 modeled structure of ARC is supported by intramolecular live-cell Förster resonance energy transfer 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. The findings have important implications for the structural organization of ARC with respect to distinct functions, such as postsynaptic signal transduction and virus-like capsid formation. Open Practices Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Arc Interacts with the Integral Endoplasmic Reticulum Protein, Calnexin.

Activity-regulated cytoskeleton-associated protein, Arc, is a major regulator of long-term synaptic plasticity and memory formation. Here we reveal a novel interaction partner of Arc, a resident endoplasmic reticulum transmembrane protein, calnexin. We show an interaction between recombinantly-expressed GST-tagged Arc and endogenous calnexin in HEK293, SH-SY5Y neuroblastoma and PC12 cells. The interaction was dependent on the central linker region of the Arc protein that is also required for endocytosis of AMPA-type glutamate receptors. High-resolution proximity-ligation assays (PLAs) demonstrate molecular proximity of endogenous Arc with the cytosolic C-terminus, but not the lumenal N-terminus of calnexin. In hippocampal neuronal cultures treated with brain-derived neurotrophic factor (BDNF), Arc interacted with calnexin in the perinuclear cytoplasm and dendritic shaft. Arc also interacted with C-terminal calnexin in the adult rat dentate gyrus (DG). After induction of long-term potentiation (LTP) in the perforant path projection to the DG of adult anesthetized rats, enhanced interaction between Arc and calnexin was obtained in the dentate granule cell layer (GCL). Although Arc and calnexin are both implicated in the regulation of receptor endocytosis, no modulation of endocytosis was detected in transferrin uptake assays. Previous work showed that Arc interacts with multiple protein partners to regulate synaptic transmission and nuclear signaling. The identification of calnexin as a binding partner further supports the role of Arc as a hub protein and extends the range of Arc function to the endoplasmic reticulum, though the function of the Arc/calnexin interaction remains to be defined.