Structural-functional analyses of the huntingtin/HAP40 complex in <i>Drosophila</i> and humans

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, interest in targeting HTT for HD treatment is growing, necessitating a clear understanding of HTT regulation. 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 <i>Drosophila</i>, controls HTT's function, protein stability, and levels, and is a potential modifier of HD pathogenesis, supporting its central role in HTT regulation. Here, we showed that HTT synergizes with HAP40 to induce novel gain-of-function effects in <i>Drosophila</i> 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 complexes, we identified ten conserved bonds that are important for HAP40's affinity for HTT. Finally, we showed that the conserved N-terminal BΦ motif in HAP40 is not essential for HTT binding but important 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 conservation of the two complexes from these evolutionarily distant species and further uncover novel insight into HAP40 regulation and its interaction with HTT.