Bustad, H. J. | Search bioRxiv

bioRxiv2021

High-affinity nanobodies as tools for structural and functional studies on mammalian Arc

Activity-regulated cytoskeleton-associated protein (Arc) is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals. However, the mechanistic and structural basis of Arc function is little understood. Arc has an NTD involved in membrane binding and a CTD which binds postsynaptic protein ligands. In addition, the NTD and CTD both function in Arc oligomerization, including assembly of retrovirus-like capsid involved in intercellular signaling. We produced and characterised six ultra-high-affinity anti-Arc nanobodies (Nb). The CTD of both rat and human Arc could be crystallised in ternary complexes with two Nbs simultaneously bound (H11 and C11). H11 binding deep into the stargazing-binding pocket of Arc CTD suggested competitive binding with Arc ligand peptides, which was confirmed in vitro. This indicates that the H11 Nb could serve as a genetically-encoded tool for inhibition of endogenous Arc N-lobe interactions in study of neuronal function and plasticity. The crystallisation of the human Arc CTD in two different conformations, accompanied by SAXS data and molecular dynamics simulations, paints a dynamic picture of the mammalian Arc CTD. Dynamics were affected by mutations known to inhibit capsid formation, implying a role for Arc CTD dynamics in oligomerisation. Dimerisation of the NTD, together with structural dynamics of the CTD, suggest a mechanism, by which structural dynamics of the CTD may promote capsomer formation, and dimerisation of the NTD links capsomers, facilitating the formation of capsids. The described recombinant ultrahigh-affinity anti-Arc Nbs are versatile tools that can be further developed for studying mammalian Arc structure and function in vitro and in vivo.

biochemistry↗

bioRxiv2019

Molecular determinants of Arc oligomerization and formation of virus-like capsids

Expression of activity-regulated cytoskeleton-associated protein (Arc) is critical for long-term synaptic plasticity, memory formation, and cognitive flexibility. The ability of Arc to self-associate and form virus-like capsid structures implies functionally distinct oligomeric states. However, the molecular mechanism of Arc oligomerization is unknown. Here, we identified a 28-amino-acid region necessary and sufficient for Arc oligomerization. This oligomerization region is located within the second coil of a predicted anti-parallel coiled-coil in the N-terminal domain (NTD). Using alanine scanning mutagenesis, we found a 7-amino-acid motif critical for oligomerization and Arc-mediated transferrin endocytosis in HEK cells. Intermolecular fluorescence lifetime imaging in hippocampal neurons confirmed self-association mediated by the motif. To quantify oligomeric size, we performed a single-molecule photobleaching analysis of purified Arc wild-type and mutant. This analysis revealed a critical role for the NTD motif in the formation of higher-order Arc oligomers (30-170 molecules). Moreover, assembly of higher-order wild-type Arc oligomers was significantly enhanced by addition of GFP RNA. Purified wild-type Arc formed virus-like capsids, as visualized by negative-stain EM, and was estimated by light scattering analysis to contain 40-55 Arc units. In contrast, mutant Arc formed a homogenous dimer population as demonstrated by single-molecule TIRF imaging, size-exclusion chromatography with multi-angle light scattering analysis, small-angle X-ray scattering analysis, and single-particle 3D EM reconstruction. Thus, the dimer appears to be the basic building block for assembly. Herein, we show that the NTD motif is essential for higher-order Arc oligomerization, assembly of virus-like capsid particles, and facilitation of oligomerization by exogenous RNA. SIGNIFICANCEArc protein is rapidly expressed in neurons in response to synaptic activity and plays critical roles in synaptic plasticity, postnatal cortical developmental, and memory. Arc has diverse molecular functions, which may be related to distinct oligomeric states of the protein. Arc has homology to retroviral Gag protein and self-assembles into retrovirus-like capsid structures that are capable of intercellular transfer of RNA. Here, we identified a motif in the N-terminal coiled-coil domain of mammalian Arc that mediates higher-order oligomerization and formation of virus-like capsids. The basic building block is the Arc dimer and exogenous RNA facilitates further assembly. The identified molecular determinants of Arc oligomerization will help to elucidate the functional modalities of Arc in the mammalian brain.

biochemistry↗