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

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. •Expression and purification of human huntingtin (HTT) subdomain protein samples•CryoEM analysis of HTT subdomain-HAP40 protein complexes•Analysis of HTT subdomain-HAP40 interactions with in vitro and in cell assays Alteen et al. designed structure-rationalized HTT subdomain constructs for structure-function interrogation of this huge protein. HTT subdomain proteins were validated using biophysical approaches and cryo-electron microscopy. Derivatized versions of these constructs were used for proof-of-principle protein interaction analyses in vitro and in cells to probe the HTT-HAP40 interaction.