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

DOI: 10.1371/journal.pone.0269281

Authors: Sigurbjörn Markússon (University of Bergen) , Erik I. Hallin (University of Bergen) , Helene J. Bustad (University of Bergen) , Arne Raasakka (University of Bergen) , Ju Xu (University of Bergen) , Gopinath Muruganandam (Vlaams Instituut voor Biotechnologie; Vrije Universiteit Brussel) , Remy Loris (Vlaams Instituut voor Biotechnologie; Vrije Universiteit Brussel) , Aurora Martinez (University of Bergen) , Clive R. Bramham (University of Bergen) , Petri Kursula (University of Bergen)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Plos One , VOL 17

State: Published (Approved)
Published: June 2022
Diamond Proposal Number(s): 18666

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.

Journal Keywords: Capsids; Crystal structure; Crystallization; Dimerization; Dimers; Small-angle scattering; Crystals; Synaptic plasticity

Subject Areas: Biology and Bio-materials, Technique Development


Instruments: I03-Macromolecular Crystallography

Other Facilities: P11 at DESY; P14 at EMBL/DESY; SWING at SOLEIL; BM29 at ESRF

Added On: 19/06/2022 10:02

Documents:
journal.pone.0269281.pdf

Discipline Tags:

Health & Wellbeing Technique Development - Life Sciences & Biotech Neurology Structural biology Life Sciences & Biotech

Technical Tags:

Macromolecular Crystallography (MX)