HAP40 orchestrates huntingtin structure for differential interaction with polyglutamine expanded exon 1

Abstract

Huntington's disease results from expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene, producing an aberrantly functioning form of HTT. Both wildtype and disease-state HTT form a hetero-dimer with HAP40 of unknown functional relevance. We demonstrate in vivo that HTT and HAP40 cellular abundance are coupled. Integrating data from a 2.6 Å cryo-electron microscopy structure, cross-linking mass spectrometry, small-angle X-ray scattering, and modeling, we provide a near-atomic-level view of HTT, its molecular interaction surfaces and compacted domain architecture, orchestrated by HAP40. Native mass-spectrometry reveals a remarkably stable hetero-dimer, potentially explaining the cellular inter-dependence of HTT and HAP40. The polyglutamine tract containing N-terminal exon 1 region of HTT is dynamic, but shows greater conformational variety in the mutant than wildtype exon 1. By providing novel insight into the structural consequences of HTT polyglutamine expansion, our data provide a foundation for future functional and drug discovery studies targeting Huntington's disease.

Purification of full-length recombinant human huntingtin proteins with allelic series of polyglutamine lengths

“…The quality of the complex was examined by SDS- and native-PAGE ( Figure 6 B). We also noticed that HTT_HAP40 complex forms a multimer, which is contrast to the previous works ( Guo et al., 2018 ; Harding et al., 2021 ; Huang et al., 2021 ). However, it is possible that the multimer of HTT_HAP40 complex may be induced in high concentration, which is in our case.…”

“…Although the purified HTT protein can be pulled down with GST-HAP40, HTT_HAP40 does not form a stable complex when compared with the complex coexpressed, which is consistent with previous works ( Guo et al., 2018 ; Harding et al., 2021 ; Huang et al., 2021 ). These data demonstrated that the HTT complexes can be generated through this protocol.…”

“…However, we have shown that the recombinant HTT purified by using this protocol activates the histone H3K27 tri-methylase activity of Polycomb Repressive Complex 2 (PRC2) in vitro ( Seong et al., 2010 ). In addition, our cryo-EM analysis (EMDB: EMD-4944) ( Jung et al., 2020 ) on full-length HTT proteins obtained using this method shows that they have similar structures to those previously resolved high-resolution cryo-EM structure of HTT ( Guo et al., 2018 ; Harding et al., 2021 ; Huang et al., 2021 ) and maintains the similar phosphorylation pattern to the previously identified phosphorylation sites ( Harding et al., 2019 ; Jung et al., 2020 ; Saudou and Humbert, 2016 ). These results suggest that this protocol to generate recombinant full-length HTT proteins with allelic series of polyQ lengths will provide key reagents for various experiments to investigate the functions of HTT and the molecular mechanism of HD pathology.…”