A Map of Protein-Metabolite Interactions Reveals Principles of Chemical Communication

Abstract

Metabolite-protein interactions control a variety of cellular processes, thereby playing a major role in maintaining cellular homeostasis. Metabolites comprise the largest fraction of molecules in cells, but our knowledge of the metabolite-protein interactome lags behind our understanding of protein-protein or protein-DNA interactomes. Here, we present a chemoproteomic workflow for the systematic identification of metabolite-protein interactions directly in their native environment. The approach identified a network of known and novel interactions and binding sites in Escherichia coli, and we demonstrated the functional relevance of a number of newly identified interactions. Our data enabled identification of new enzyme-substrate relationships and cases of metabolite-induced remodeling of protein complexes. Our metabolite-protein interactome consists of 1,678 interactions and 7,345 putative binding sites. Our data reveal functional and structural principles of chemical communication, shed light on the prevalence and mechanisms of enzyme promiscuity, and enable extraction of quantitative parameters of metabolite binding on a proteome-wide scale.

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

“…The lower number of detected interactions in the current dataset is likely due to a combination of (i) a new assay buffer with physiological salt concentrations, to minimize unspecific binding; and (ii) more stringent analysis, including fully automated peak picking and exclusion of metabolite signals that are unstable over time. Our experiments included 27 proteins and 18 metabolites that were also covered in a recent proteomic study based on limited proteolysis coupled to mass spectrometry (LiP‐MS; Piazza et al , ). The overlap between both studies was 11 interactions, six of which were not reported before, similarly to what can be expected from other large‐scale studies (Diether & Sauer, ).…”

“…These differences could result from NMR reporting on direct interactions under in vitro conditions, while LiP‐MS senses both direct and indirect effects under native cellular extract conditions. Furthermore, while T1rho NMR is most sensitive to interactions with μM dissociation constants (Nikolaev et al , ), LiP‐MS exhibits a broader sensitivity range (Piazza et al , ). Despite the many non‐overlapping interactions, the amount of interactions detected for the same metabolites was comparable (correlation with R 2 = 0.2857; Appendix Fig S11).…”

PROMIS: Global Analysis of PROtein‐Metabolite Interactions

“…Alternative strategies rely on biochemical methods and can be divided into three groups: (i) ligand‐targeted, in which a metabolite is used as bait to determine the targets of the small molecule; (ii) protein‐targeted, which aim to identify small molecules affecting the function of a signaling protein; and (iii) untargeted, which chart a map of the protein–metabolite interactome in a given organism (Luzarowski & Skirycz, ). Examples of ligand‐targeted approaches include classical strategies like affinity purification, in which a chemical compound is immobilized to the matrix and used to capture interacting proteins from native cellular lysate, as well as the recent methods TPP/CETSA, SPROX, and LiP‐SMAP, which monitor changes in the protein properties caused by ligand binding (Kosmacz et al., ; Ong et al., ; Piazza et al., ; Savitski et al., ; West, Tang, & Fitzgerald, ). These properties can be thermal stability of a protein, oxidation rate, or susceptibility of a protein to proteolysis.…”

“…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., ).…”

“…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., ). PROMIS, however, takes advantage of the recent progress in mass spectrometry–based proteomics and metabolomics and enables analysis of the elution profiles of thousands of endogenous proteins and metabolites in a single experiment without the need to modify either proteins or metabolites.…”