Partners in Piwi: AlphaFold-Based Structural Screening in Drosophila

Abstract

Piwi is a PIWI-clade Argonaute protein that functions in transposon silencing and germline stem cell regulation in Drosophila melanogaster, but the structural basis of many Piwi protein–protein interactions remains unclear. This study used a computational screening strategy to identify candidate Piwi interaction interfaces by combining FlyBase Gene Ontology-based candidate selection with AlphaFold- and ColabFold-based complex prediction. Piwi-centered multimer predictions were ranked using pLDDT, predicted aligned error (PAE), predicted template modeling score (pTM), and interface predicted template modeling score (ipTM), and top-ranked models were further analyzed in ChimeraX through structural superposition, RMSD comparison, and sequence alignment against known experimental Piwi structures. Most candidate complexes showed low-to-moderate interface confidence, with only a small subset emerging as stronger structural candidates. Comparisons with experimental structures demonstrated that the workflow reliably recovered major features of the Piwi core, supporting its use as a proof-of-concept method for structural screening. Candidate complexes nevertheless varied substantially in interface confidence: Piwi–Panx showed weak support for a stable interface, whereas Piwi–Arx emerged as a stronger candidate complex with higher overall confidence. These results show that AlphaFold/ColabFold-based screening can prioritize candidate Piwi partners and structural interfaces for future validation, providing a foundation for downstream mutagenesis and biochemical analysis of Piwi function.