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

Item Type:

Article

Title:

Creators Name:

Alteen, M.G. and Deme, J.C. and Alvarez, C.P. and Loppnau, P. and Hutchinson, A. and Seitova, A. and Chandrasekaran, R. and Silva Ramos, E. and Secker, C. and Alqazzaz, M. and Wanker, E.E. and Lea, S.M. and 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:

Number:

Page Range:

1121-1131.e6

Date:

7 September 2023

Official Publication:

https://doi.org/10.1016/j.str.2023.06.002

PubMed:

View item in PubMed

Related to:

URL	URL Type
https://edoc.mdc-berlin.de/22854/	Preprint version

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