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

Area of Science:

• Biochemistry
• Systems Biology
• Structural Biology

Background:

• Enzymes in metabolic pathways often form metabolons via weak protein-protein interactions (PPI) to localize and protect metabolites.
• The transient nature of these enzyme assemblies has made their structural architecture difficult to determine, hindering efforts to engineer new metabolic pathways.

Purpose of the Study:

• To create a comprehensive protein-protein interaction (PPI) map for the E. coli 1-carbon metabolism pathway.
• To elucidate the structural and biophysical principles governing metabolon formation and function.

Main Methods:

• Utilized bimolecular fluorescence complementation (BiFC) for in vivo detection of transient PPIs.
• Employed scanning mutagenesis, AlphaFold predictions, and metadynamics simulations to analyze protein interfaces.
• Conducted diffusion-reaction simulations to model pathway flux.

Main Results:

• Generated a PPI map of 1225 interactions in E. coli 1-carbon metabolism, identifying significant clustering within folate and purine biosynthesis pathways.
• Discovered that proteins predominantly use conserved, active site-distant interfaces for interactions with multiple partners.
• Demonstrated a substantial increase in metabolic pathway flux through simulations incorporating realistic PPI networks and shared interaction surfaces.

Conclusions:

• This study provides fundamental insights into the biophysics and structural characteristics of metabolons and transient binary complexes.
• The findings highlight the role of specific PPIs in enhancing metabolic efficiency and offer a framework for re-engineering metabolic pathways.