PolyQ expansion does not alter the Huntingtin-HAP40 complex

Huang, Bin; Guo, Qiang; Ml, Niedermeier; Cheng, Jin Q.; Engler, Tatjana; Maurer, Melanie; Pautsch, Alexander; Baumeister, Wolfgang; Stengel, Florian; Kochanek, Stefan; Fernández-Busnadiego, Rubén

doi:10.1101/2021.02.02.429316

Cited by 2 publications

(2 citation statements)

References 32 publications

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“…High-resolution structure of HTT-HAP40 Complex HTT-HAP40 was expressed in insect cells and purified as previously described 21 . We determined the structure of HTT-HAP40 (PDBID: 6X9O) to a nominal resolution of 2.6 Å using cryo-EM (Figure 2a, Figure 2b and Supplementary Figure 1), improving substantially upon the previously published 4 Å model (PDBID: 6EZ8; Guo et al, 2018) and two recently deposited models (PDBIDs: 7DXJ [3.6 Å] and 7DKK [4.1 Å]; Huang et al, 2021). Similar to all previous models, flexible regions accounting for ~25% of the HTT-HAP40 complex, including exon 1 and the IDR, were not resolved in our high-resolution maps (Figure 2c).…”

Section: Resultsmentioning

confidence: 96%

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

Harding

Deme

Hevler

et al. 2021

Preprint

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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.

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Section: Resultsmentioning

confidence: 96%

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

Harding

Deme

Hevler

et al. 2021

Preprint

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“…The structure of HTT has been solved by cryo-electron microscopy (Guo et al, 2018; Harding et al, 2021; Huang et al, 2021a), in complex with 40 kDa huntingtin-associated protein (HAP40) (Peters and Ross, 2001), forming a ~ 389 kDa multidomain complex. HTT is primarily composed of HEAT repeats ( h untingtin, e longation factor 3, protein phosphatase 2 a , and yeast kinase T OR1) (Andrade et al, 2001) that form a large solenoid-like structure at the N-terminal region of the protein as well as a more compact C-terminal region.…”

Section: Introductionmentioning

confidence: 99%

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

Alteen

Deme

Alvarez

et al. 2022

Preprint

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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.

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PolyQ expansion does not alter the Huntingtin-HAP40 complex

Cited by 2 publications

References 32 publications

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

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

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

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