Abstract

Huntington’s disease (HD) is a neurodegenerative disorder caused by an abnormal CAG expansion in the Huntingtin (HTT) gene. Given its simple genetic cause but complex pathogenic mechanisms, inter- est in targeting HTT for HD treatment is growing, necessitating a clear understanding of HTT regula- tion. HTT protein primarily exists in a core complex with HAP40, forming a highly ordered structure with two large globular domains connected by a bridge. We previously demonstrated that HAP40 is conserved in Drosophila, controls HTT’s function, protein stability, and levels, and is a potential modi- fier of HD pathogenesis, supporting its central role in HTT regulation. Here, we showed that HTT syner- gizes with HAP40 to induce novel gain-of-function effects in Drosophila when overexpressed. Protein modeling revealed that despite their prominent evolutionary and sequence divergence, the fly and human HTT-HAP40 complexes share a high degree of structural similarity. Protein-contact maps and molecular simulations showed that HAP40 preferentially binds to HTT’s C-terminal domain in both complexes. By examining the interfacial contacts between HTT and HAP40 in fly and human com- plexes, we identified ten conserved bonds that are important for HAP40’s affinity for HTT. Finally, we showed that the conserved N-terminal BU motif in HAP40 is not essential for HTT binding but impor- tant for HAP40’s functions. Through the structural-functional analyses of the fly and human HTT- HAP40 complexes, our results support that the structural similarity underlies the functional conserva- tion of the two complexes from these evolutionarily distant species and further uncover novel insight into HAP40 regulation and its interaction with HTT.