A comprehensive map of transient protein-protein interactions and their structural features in theE. coli1-carbon metabolism pathway

Abstract

Enzymes in a pathway often form metabolons through weak protein-protein interactions (PPI) that localize and protect labile metabolites. Due to their transient nature, the structural architecture of these enzyme assemblies has largely remained elusive. Here we delineate a complete PPI map of theE. coli1-carbon metabolism pathway using bimolecular fluorescence complementation that can detect transient interactionsin vivoand show strong intra- and inter-pathway clusters within the folate and purine biosynthesis pathways. Using deep scanning mutagenesis we identify the PPI interface on DHFR. Further, a combination of AlphaFold predictions with meta-dynamics simulations reveal interaction interfaces of all PPI in the pathway, which not only agrees with experimental findings but also reveal that most proteins use conserved “dedicated” interfaces distant from their active sites to interact with multiple partners. Overall, our data reveals the structural architecture of binary PPI complexes in a metabolon in a crucial prokaryotic metabolic pathway.