AlphaFold‐driven discovery of oxysterol‐binding protein <scp>‐</scp> related protein‐phosphoinositide 3‐, 4‐, and 5‐phosphatase interactions using new generation confidence scores

Non-vesicular lipid transport contributes to the regulation of membrane composition and organelle function at membrane contact sites. OSBP-related proteins (ORPs) are central to this process, yet their interaction networks remain incompletely defined. Here, we systematically screened potential interactions between ORPs and phosphoinositide 3-, 4-, and 5-phosphatases using AlphaPulldown2, AlphaFold2-Multimer, and AlphaFold3. We established a protocol for model generation by combining AlphaFold2-Multimer predictions (including five-replicates) with an AlphaPulldown2 interaction screen across around 200 protein pairs, and with AlphaFold3 predictions including lipid-bound and multimeric assemblies. Interface confidence was assessed for consistency using the weighted ipTM + pTM metric, actifpTM, new generation ipSAE scoring, and FoldSeek-Multimer clustering. We further evaluated the protein pairs' biological plausibility based on subcellular localization data, in silico membrane insertion, evolutionary conservation via ConSurf, and protein binding interface analysis using the deep learning tool PeSTo. This integrative protocol uncovered functionally conserved binding modes in the SAC1 lipid phosphatase with the ORP family, particularly with ORP11, and predicted functionally relevant protein-lipid interfaces.