Markússon S, Hallin EI, Bustad HJ, Raasakka A, Xu J, Muruganandam G, et al. (2022) High-affinity anti-Arc nanobodies provide tools for structural and functional studies. PLoS ONE 17(6): e0269281. https://doi.org/10.1371/journal.pone.0269281
High-affinity anti-Arc nanobodies provide tools for structural and functional studies
|Author:||Markússon, Sigurbjörn1; Hallin, Erik I.1; Bustad, Helene J.1;|
1Department of Biomedicine, University of Bergen, Bergen, Norway
2VIB-VUB Center for Structural Biology, Vlaams Instituut voor Biotechnologie, Brussels, Belgium
3Department of Bioengineering Sciences, Structural Biology Brussels, Vrije Universiteit Brussel, Brussel, Belgium
4Faculty of Biochemistry and Molecular Medicine & Biocenter Oulu, University of Oulu, Oulu, Finland
|Online Access:||PDF Full Text (PDF, 5.9 MB)|
|Persistent link:|| http://urn.fi/urn:nbn:fi-fe2023022228301
Public Library of Science,
|Publish Date:|| 2023-02-22
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.
|Type of Publication:||
A1 Journal article – refereed
|Field of Science:||
1182 Biochemistry, cell and molecular biology
The refined coordinates and structure factors were deposited at the Protein Data Bank with entry codes 7R20 (Nb E5), 7R24 (rArc-CTD complex), 7R23 (extended hArc-CTD complex), and 7R1Z (collapsed hArc-CTD complex). All further relevant data are within the manuscript and its Supporting information files.
© 2022 Markússon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.