Systematic mapping of protein‐metabolite interactions in central metabolism of Escherichia coli

Abstract

Metabolite binding to proteins regulates nearly all cellular processes, but our knowledge of these interactions originates primarily from empirical in vitro studies. Here, we report the first systematic study of interactions between water‐soluble proteins and polar metabolites in an entire biological subnetwork. To test the depth of our current knowledge, we chose to investigate the well‐characterized Escherichia coli central metabolism. Using ligand‐detected NMR , we assayed 29 enzymes towards binding events with 55 intracellular metabolites. Focusing on high‐confidence interactions at a false‐positive rate of 5%, we detected 98 interactions, among which purine nucleotides accounted for one‐third, while 50% of all metabolites did not interact with any enzyme. In contrast, only five enzymes did not exhibit any metabolite binding and some interacted with up to 11 metabolites. About 40% of the interacting metabolites were predicted to be allosteric effectors based on low chemical similarity to their target's reactants. For five of the eight tested interactions, in vitro assays confirmed novel regulatory functions, including ATP and GTP inhibition of the first pentose phosphate pathway enzyme. With 76 new candidate regulatory interactions that have not been reported previously, we essentially doubled the number of known interactions, indicating that the presently available information about protein–metabolite interactions may only be the tip of the iceberg.

Label-free quantitative proteomic analysis of the inhibition effect of Lactobacillus rhamnosus GG on Escherich coli biofilm formation in coculture

“…Proteomics analysis revealed that coculture with E.coli signi cantly up-regulated two nucleotide metabolism-related genes, purD and purM. The pur-operon (purEKCSQLFMNHD) is responsible for the catalysis of de novo synthesis of inosine monophosphate (IMP) from phosphoribosyl pyrophosphate [19]. In S. aureus, purine biosynthesis enzymes have been closely implicated in virulence, persistence, and tolerance to stresses such as antibiotic resistance [20] [21].…”

PROMIS: Global Analysis of PROtein‐Metabolite Interactions

“…interactions in biological systems remains unknown (Diether, Nikolaev, Allain, & Sauer, 2019;Gallego et al, 2010;Li, Gianoulis, Yip, Gerstein, & Snyder, 2010;Piazza et al, 2018). Most currently available methods are limited by the availability of a recombinant protein, exploit compound libraries rather than complex metabolite extracts, or focus on a single, preselected small molecule Luzarowski & Skirycz, 2019;Maeda et al, 2013;Piazza et al, 2018;Savitski et al, 2014).…”

“…PROtein–metabolite interactions using size separation (PROMIS) is a biochemical method for studying protein–small molecule interactions on a proteome‐ and metabolome‐wide scale, based on the simple assumption that the small molecules or molecular complexes will exhibit similar chromatographic behavior (Veyel et al., , ). Despite a few systematic studies, the true extent of protein–small molecule interactions in biological systems remains unknown (Diether, Nikolaev, Allain, & Sauer, ; Gallego et al., ; Li, Gianoulis, Yip, Gerstein, & Snyder, ; Piazza et al., ). Most currently available methods are limited by the availability of a recombinant protein, exploit compound libraries rather than complex metabolite extracts, or focus on a single, preselected small molecule (Luzarowski et al., ; Luzarowski & Skirycz, ; Maeda et al., ; Piazza et al., ; Savitski et al., ).…”

The Regulatory Role of Key Metabolites in the Control of Cell Signaling

“…Therefore, some molecular events should connect given metabolic reactions with signal transduction cascades, in order to coherently integrate a given flux distribution with the activity of regulatory proteins, such as the kinases upstream to signaling pathways. However, recent publications support the idea that we still need more data to understand the role of specific metabolites in controlling proteins activity at a systems level [ 7 , 8 ].…”

“…On the contrary, the systematic study of the interaction between proteins and metabolites (PMI, protein-metabolite interaction) has started to be explored only very recently. Although recent reports suggest an important role of PMIs in the regulation of signaling [ 9 , 10 ], most publications still focus on the effect of the metabolites belonging to glycolysis and TCA cycle on the activity of the enzymes of the same pathways [ 7 , 8 , 11 ]. Therefore, a systematic analysis of the interaction between molecules of the central carbon metabolism and the proteins that compose the signal transduction network is still largely missing.…”