Delineation of functional subdomains of Huntingtin protein and their interaction with HAP40

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Item Type:	Article
Title:	Delineation of functional subdomains of Huntingtin protein and their interaction with HAP40
Creators Name:	Alteen, M.G., Deme, J.C., Alvarez, C.P., Loppnau, P., Hutchinson, A., Seitova, A., Chandrasekaran, R., Silva Ramos, E., Secker, C., Alqazzaz, M., Wanker, E.E., Lea, S.M., Arrowsmith, C.H. and Harding, R.J.
Abstract:	The huntingtin (HTT) protein plays critical roles in numerous cellular pathways by functioning as a scaffold for its many interaction partners and HTT knock out is embryonic lethal. Interrogation of HTT function is complicated by the large size of this protein so we studied a suite of structure-rationalized subdomains to investigate the structure-function relationships within the HTT-HAP40 complex. Protein samples derived from the subdomain constructs were validated using biophysical methods and cryo-electron microscopy, revealing they are natively folded and can complex with validated binding partner, HAP40. Derivatized versions of these constructs enable protein-protein interaction assays in vitro, with biotin tags, and in cells, with luciferase two-hybrid assay-based tags, which we use in proof-of-principle analyses to further interrogate the HTT-HAP40 interaction. These open-source biochemical tools enable studies of fundamental HTT biochemistry and biology, will aid the discovery of macromolecular or small-molecule binding partners and help map interaction sites across this large protein.
Keywords:	Huntington’s Disease, Neurodegeneration, Protein Purification, Protein Structure, Cryo-Electron Microscopy, Protein-Protein Interaction
Source:	Structure
ISSN:	0969-2126
Publisher:	Cell Press
Volume:	31
Number:	9
Page Range:	1121-1131.e6
Date:	7 September 2023
Additional Information:	Copyright © 2023 Elsevier Ltd. All rights reserved.
Official Publication:	https://doi.org/10.1016/j.str.2023.06.002
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