Conserved interfaces mediate multiple protein-protein interactions in a prokaryotic metabolon

Area of Science:

• Biochemistry
• Structural Biology
• Systems Biology

Background:

• Enzymes in metabolic pathways can form metabolons via weak protein-protein interactions (PPI).
• These enzyme assemblies localize and protect unstable metabolites, but their transient nature makes structural analysis challenging.
• Understanding metabolon architecture is crucial for re-engineering metabolic pathways.

Purpose of the Study:

• To create a comprehensive protein-protein interaction (PPI) map for the E. coli 1-carbon metabolism pathway.
• To investigate the structural basis and functional implications of enzyme complex formation.
• To elucidate the biophysical principles governing metabolon organization and dynamics.

Main Methods:

• Utilized bimolecular fluorescence complementation (BiFC) for in vivo capture of transient PPIs.
• Employed scanning mutagenesis, AlphaFold predictions, and meta-dynamics simulations for structural analysis.
• Conducted diffusion-reaction simulations to model pathway flux with realistic PPI networks.

Main Results:

• Generated a map of 1225 PPIs within the E. coli 1-carbon metabolism pathway.
• Identified significant intra- and inter-pathway clustering, particularly in folate and purine biosynthesis.
• Revealed that proteins commonly use conserved, active site-distant interfaces for multiple interactions.
• Demonstrated a substantial increase in metabolic pathway flux due to shared interaction surfaces and PPI networks.

Conclusions:

• Metabolon formation is governed by conserved, dedicated protein interfaces.
• Transient enzyme complexes significantly enhance metabolic efficiency.
• This study provides fundamental insights into metabolon biophysics and the structural basis of transient enzyme interactions.