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

Abstract

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 poorly understood. Arc has an N-terminal domain (NTD) involved in membrane binding and a C-terminal domain (CTD) that binds postsynaptic protein ligands. In addition, the NTD and CTD both function in Arc oligomerisation, including assembly of retrovirus-like capsids involved in intercellular signalling. To obtain new tools for studies on Arc structure and function, we produced and characterised six high-affinity anti-Arc nanobodies (Nb). The CTD of rat and human Arc were both crystallised in ternary complexes with two Nbs. One Nb bound deep into the stargazin-binding pocket of Arc CTD and suggested competitive binding with Arc ligand peptides. 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. The collapsed conformation closely resembles Drosophila Arc in capsids, suggesting that we have trapped a capsid-like conformation of the human Arc CTD. Our data obtained with the help of anti-Arc Nbs suggest that structural dynamics of the CTD and dimerisation of the NTD may promote the formation of capsids. Taken together, the recombinant high-affinity anti-Arc Nbs are versatile tools that can be further developed for studying mammalian Arc structure and function, as well as mechanisms of Arc capsid formation, both in vitro and in vivo. For example, the Nbs could serve as a genetically encoded tools for inhibition of endogenous Arc interactions in the study of neuronal function and plasticity.

Molecular physiology of Arc/Arg3.1: The oligomeric state hypothesis of synaptic plasticity

“…Tools that would allow labeling or functional manipulation of endogenous Arc would be an advantage. To this end, single‐domain antibodies (nanobodies) that bind the Arc CA domain have been developed 138,139 . The recombinant nanobodies bind with high affinity (low nM Kd) and are suitable for expression in mammalian cells as intra‐bodies.…”

“…To this end, single‐domain antibodies (nanobodies) that bind the Arc CA domain have been developed. 138 , 139 The recombinant nanobodies bind with high affinity (low nM Kd) and are suitable for expression in mammalian cells as intra‐bodies. One of the nanobodies, ArcNb H11, binds selectively to the N‐lobe ligand pocket with 35 000 times higher affinity than stargazin, competitively inhibits stargazin binding, and allows affinity purification of intra‐cellular Arc.…”

Structural characterization of two nanobodies targeting the ligand-binding pocket of human Arc

“…The high-resolution structure of full-length Arc (flArc) remains unknown, as the molecular flexibility of Arc hinders its crystallization [36]. To face this problem, anti-Arc nanobodies have been developed and used as crystallization chaperones for the rat and human Arc C-terminal domain (Arc-CTD) [37]. Isothermal titration calorimetry (ITC) revealed that the anti-Arc nanobody H11 can displace a TARPγ2-derived peptide from the multi-peptide binding pocket of flArc, suggesting a role for this nanobody as a functional modulator of Arc function [37].…”

“…To face this problem, anti-Arc nanobodies have been developed and used as crystallization chaperones for the rat and human Arc C-terminal domain (Arc-CTD) [37]. Isothermal titration calorimetry (ITC) revealed that the anti-Arc nanobody H11 can displace a TARPγ2-derived peptide from the multi-peptide binding pocket of flArc, suggesting a role for this nanobody as a functional modulator of Arc function [37]. This finding has prompted an increased interest in anti-Arc nanobodies that selectively bind Arc-NL, as it mediates several essential functions for the regulation of synaptic plasticity, and as the binding event could affect full-length Arc conformation and oligomeric state.…”

“…Both nanobodies bound to the multi-peptide binding pocket of Arc-NL, which validates them as possible functional modulators of Arc-NL. Nanobody E5 bound flArc simultaneously with nanobody C11, known to target the Arc C-lobe [37, 38]. These findings open a path towards using anti-Arc-NL nanobodies to characterize the different structural states and complex molecular functions of the Arc protein.…”