# Conserved interfaces mediate multiple protein–protein interactions in a prokaryotic metabolon

## TL;DR

The study reveals how enzymes in E. coli's 1-carbon metabolism form transient clusters using conserved interfaces, which helps speed up metabolic reactions.

## Contribution

The paper identifies conserved protein interfaces used for multiple transient interactions in a prokaryotic metabolon, revealing how they enhance metabolic flux.

## Key findings

- A PPI map of 1225 interactions in E. coli's 1-carbon metabolism was created using bimolecular fluorescence complementation.
- Enzymes use conserved interfaces distant from active sites to interact with multiple partners.
- Shared interfaces increase reaction fluxes by several orders of magnitude.

## 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, limiting our abilities to re-engineer novel metabolic pathways. Here, we delineate a complete PPI map of 1225 interactions in the E. coli 1-carbon metabolism pathway using bimolecular fluorescence complementation that can capture transient interactions in vivo and show strong intra- and inter-pathway clusters within the folate and purine biosynthesis pathways. Scanning mutagenesis experiments along with AlphaFold predictions and metadynamics simulations reveal that most proteins use conserved “dedicated” interfaces distant from their active sites to interact with multiple partners. Diffusion-reaction simulations with shared interaction surfaces and realistic PPI networks reveal a dramatic speedup in metabolic pathway fluxes. Overall, this study sheds light on the fundamental features of metabolon biophysics and structural aspects of transient binary complexes.

A map of transient and weak interactions among members of the E. coli 1-carbon metabolon is delineated, and enzymes that use a conserved dedicated interface to engage in such PPI are uncovered. Such complexes speed up enzymatic reactions and fluxes by several orders of magnitude.

The folate pathway with the most labile metabolites shows strongest cluster formation, while the purine pathway also shows moderate interactions along with strong structural similarity.Interacting proteins in the 1-carbon metabolism pathway behave like singlish or date hubs, as opposed to multi or party hubs.Shared interfaces might be evolutionarily important to maintain the dynamic nature of metabolons, while also posing a natural limit on the size of the metabolon.Such enzyme clusters enhance reaction fluxes while individual enzymes could operate far from the diffusion limit.This study adds to the list of a few known examples of metabolon formation in bacterial systems, again indicating presence of a considerable degree of self-organization in prokaryotes.

The folate pathway with the most labile metabolites shows strongest cluster formation, while the purine pathway also shows moderate interactions along with strong structural similarity.

Interacting proteins in the 1-carbon metabolism pathway behave like singlish or date hubs, as opposed to multi or party hubs.

Shared interfaces might be evolutionarily important to maintain the dynamic nature of metabolons, while also posing a natural limit on the size of the metabolon.

Such enzyme clusters enhance reaction fluxes while individual enzymes could operate far from the diffusion limit.

This study adds to the list of a few known examples of metabolon formation in bacterial systems, again indicating presence of a considerable degree of self-organization in prokaryotes.

A map of transient and weak interactions among members of the E. coli 1-carbon metabolon is delineated, and enzymes that use a conserved dedicated interface to engage in such PPI are uncovered. Such complexes speed up enzymatic reactions and fluxes by several orders of magnitude.