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 antiparallel 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.

Characterization of the C-terminal tail of the Arc protein

“…Taken together, these data suggest that the C-terminal tail has minimum (if any) interaction with the N-lobe and C-lobe (together they form the capsid domain), and it does not affect the folding of the capsid domain. This observation agrees with a previous finding that the C-terminal tail lies outside the core of the Arc protein, which is consisted of the Arc N-terminus sitting above the capsid domain [19]. However, further experiments are required to examine the possible interaction between the C-terminal tail and the capsid domain.…”

“…They found that Arc’s coiled-coil subdomain (Arc 26-130 ) lies above its bi-lobe subdomain (Arc 210-361 ), and its N- and C-terminal tails lie at opposite ends. They also found that ligand binding to Arc’s N-lobe did not cause major conformational changes to the rest of the protein [ 19 ]. Furthermore, Nielsen et al produced the capsid domain of rat Arc (Arc 206-364 ) that includes the N-lobe and the C-lobe, and solved its structure using the solution NMR method [ 20 ].…”

Crystal and solution structures reveal oligomerization of individual capsid homology domains of Drosophila Arc

“…The details of the protein constructs are given in S1 Table. The expression and purification of human Arc NL and CL have been described [17].…”

“…The Arc-NT is predicted to have homology to the retroviral matrix domain and is required for the formation of large mArc oligomers. Without its N-terminal domain, mArc is monomeric in solution [17]. In mArc, it is likely that the presence of both mArc-NT and mArc-CT are required for high-order oligomerization and capsid formation [18,19].…”

“…T h i s s h o u l d t h e n b e r e p e a t e d s t a r t i n g f r o m t h e N L . U n f o r t u n a t e [17,18]. T h e r e f o r e , o l i g o m e r i z a t i o n o f t h e d A r c l o b e s l i k e l y r e p r e s e n t s f u n c t i o n a l c o m p e n s a t i o n f o r t h e l a c k o f t h e N -t e r m i n a l d o m a i n .…”