Expanding the Huntington’s disease research toolbox; validated subdomain protein constructs for biochemical and structural investigation of huntingtin

Abstract

Huntington's disease is characterised by the CAG expansion in the huntingtin gene above a critical threshold of ~35 repeats, which results in the polyglutamine expansion of the huntingtin protein (HTT). The biological roles of wildtype HTT and the associated mechanisms of disease pathology caused by expanded HTT remain incompletely understood, in part due to challenges in directly characterising interactions between HTT and putative binding partners. Here we describe a biochemical toolkit of two rationally designed, high-quality recombinant HTT subdomains; one consisting of the N-terminal HEAT region and bridge domain (NTD) and the second comprising the C-terminal HEAT domain (CTD). Using biophysical methods and cryo-electron microscopy, we show that these smaller subdomains are natively folded and can associate to reconstitute a functional full-length HTT structure capable of forming a near native-like complex with the 40 kDa HTT-associated protein (HAP40). We also report biotin-tagged variants of these subdomains, as well as full-length HTT, that permit immobilisation of each protein for quantitative biophysical assays without impacting protein quality. We demonstrate that the CTD alone can form a stable complex when co-expressed with HAP40, which can be structurally resolved. The CTD/HAP40 complex binds to the NTD, with a dissociation constant of approximately 10 nM as measured by bio-layer interferometry. We validated the interaction between the CTD and HAP40 in cells using a luciferase two-hybrid assay and used individual subdomain constructs to demonstrate their respective stabilization of HAP40 in cells. These open-source biochemical tools will prove useful to the wider HD community for the study of fundamental HTT biology, discover new macromolecular or small-molecule binding partners and mapping of their interaction sites across this very large protein.

Poly ADP-Ribose Signaling is Dysregulated in Huntington Disease

“…It should be noted that while these results support a direct interaction, the technical constraints of the experiment preclude saturation of the binding curve and therefore determination of binding kinetics and specificity. We next tested the PAR binding ability of different huntingtin subdomain constructs [78], and found that a construct comprising the N-HEAT and Bridge domains, which contains PBM-3, binds 26-mer PAR, while the C-HEAT domain (in complex with HAP40) does not ( Fig 5B ). We did not detect a significant difference in PAR binding between wild type and mutant huntingtin, nor upon deletion of the exon 1 domain in this assay ( Fig 5B ).…”