Site-resolved spatial and structural interactome of a human cell

The spatial and structural organization of proteins plays a crucial role in virtually every process within human cells. To systematically map this cellular proteome architecture, researchers employed a combination of gentle subcellular fractionation through differential ultracentrifugation and cross-linking mass spectrometry. This approach yielded 164,146 residue-to-residue links in HEK293 cells, with a resolution fine enough to pinpoint protein localizations at the sub-organelle level, determine protein orientations within cellular membranes, and identify inter-organelle contact sites.

These detailed insights translated into the discovery of 18,074 direct protein-protein interactions (PPIs), which were then integrated into AlphaFold-based pipelines to identify PPI-mediating short linear motifs and generate assembly models of large protein complexes. By leveraging these spatial and structural readouts, researchers were able to uncover new PPIs within the endomembrane system, shedding light on how they regulate the compartmental localization of trafficking machinery.

To further enhance their findings, the research team expanded their HEK293 dataset by incorporating PPI data from various cell lines and methodologies. This network topology-driven strategy allowed them to augment the spatial and structural interactome of human cells, providing a more comprehensive understanding of protein interactions in different cellular contexts.