Systematic Identification of Protein–Metabolite Interactions in Complex Metabolite Mixtures by Ligand-Detected Nuclear Magnetic Resonance Spectroscopy
Abstract
Protein-metabolite interactions play a vital role in the regulation of numerous cellular processes. Consequently, identifying such interactions is a key prerequisite for understanding cellular regulation. However, the noncovalent nature of the binding between proteins and metabolites has so far hampered the development of methods for systematically mapping protein-metabolite interactions. The few available, largely mass spectrometry-based, approaches are restricted to specific metabolite classes, such as lipids. In this study, we address this issue and show the potential of ligand-detected nuclear magnetic resonance (NMR) spectroscopy, which is routinely used in drug development, to systematically identify protein-metabolite interactions. As a proof of concept, we selected four well-characterized bacterial and mammalian proteins (AroG, Eno, PfkA, and bovine serum albumin) and identified metabolite binders in complex mixes of up to 33 metabolites. Ligand-detected NMR captured all of the reported protein-metabolite interactions, spanning a full range of physiologically relevant Kd values (low micromolar to low millimolar). We also detected a number of novel interactions, such as promiscuous binding of the negatively charged metabolites citrate, AMP, and ATP, as well as binding of aromatic amino acids to AroG protein. Using in vitro enzyme activity assays, we assessed the functional relevance of these novel interactions in the case of AroG and show that l-tryptophan, l-tyrosine, and l-histidine act as novel inhibitors of AroG activity. Thus, we conclude that ligand-detected NMR is suitable for the systematic identification of functionally relevant protein-metabolite interactions.
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NMR spectroscopy has a long-standing history of being used to effectively detect and measure a wide range of protein-ligand interactions, applicable in high-throughput fashion.
“An example of the employment of NMR in functional analysis is the detection of protein–metabolite interactions, in fact NMR spectroscopy has a long-standing history of being used to effectively detect and measure a wide range of protein–ligand interactions,144–146 which is also applicable in a high-throughput fashion.”
From Connecting metabolome and phenotype: recent advances in functional metabolomics tools for the identification of bioactive natural products (2024) · cites this paper as [146]
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- Authors
- Nikolaev, Y. V., Kochanowski, K., Link, H., Sauer, U., & Allain, F. H.-T.
- Affiliations
- ETH Zurich (CH)
- Institute of Molecular Biology & Biophysics, ETH Zürich, CH-8093 Zürich, Switzerland
- Corresponding author
- Nikolaev, Yaroslav V.
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2016-04-25
- Volume
- 55
- Issue
- 18
- Pages
- 2590-2600
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W2344146932
- Cited by
- 26
- References
- 68
- Citations by year
- 2025: 2 · 2024: 2 · 2023: 3 · 2022: 5 · 2021: 2 · 2020: 1 · 2019: 3 · 2018: 4 · 2017: 4
- FWCI
- 1.83
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Max Planck Institute for Plasma Physics (submittedVersion)
- MPG.PuRe (Max Planck Society) (submittedVersion)
- Funders
- Promedica Stiftung (1300/M)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced NMR Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Interaction Studies and Fluorescence Analysis
- Keywords
- protein-metabolite interactions, aroG, Eno, bovine serum albumin, promiscuous binding, citrate, AMP, ATP, tryptophan, tyrosine, histidine, enzyme inhibition, binding affinity, bacterial proteins, mammalian proteins
- MeSH terms
- Adenosine Monophosphate, Adenosine Triphosphate, Animals, Cattle, Serum Albumin, Bovine, Nuclear Magnetic Resonance, Biomolecular, Amino Acids, Aromatic, Escherichia coli Proteins
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