Proteome-Wide Identification and Membrane Positioning of Single-Pass Membrane Proteins Using AlphaFold Models

Abstract Single-pass (bitopic) transmembrane (TM) proteins are highly abundant and functionally diverse, yet their structures remain poorly characterized. Experimental structures of full-length bitopic proteins and their functional complexes are scarce. Although thousands of predicted protein structures from hundreds of proteomes are available, accurately identifying bitopic proteins and determining their orientations within a lipid bilayer remain challenging. Here we developed AlphaPPM, a new pipeline and web server that identifies bitopic proteins from AlphaFold (AF) models and positions them in membranes. Built on the established Positioning of Proteins in Membranes (PPM) and Folding of Membrane-Associated Peptides (FMAP) approaches, AlphaPPM automatically removes low-confidence unstructured regions and signal sequences, separates structural domains, and then reassembles and orients extracellular, intracellular, and TM domains relative to membrane boundaries. We applied and validated the method across complete sets of bitopic proteins from 18 organisms. The results are available through MembranomeX, a dedicated web resource comprising an online database and four web servers. Supplemented by manual curation and cross-validation, the database contains membrane-oriented AF models of 16,697 bitopic proteins organized into 15 functional classes, 664 superfamilies, and 2497 families and data on 7966 protein complexes. Notably, the workflow identified 1225 bitopic proteins absent from established databases. MembranomeX integrates tools for structural modeling, data retrieval, model verification, and 3D visualization, providing a comprehensive platform for evolutionary, structural, and functional analyses of bitopic proteins.