Systematic identification of protein-metabolite interactions in complex metabolite mixtures by ligand-detected NMR spectroscopy.
Systematic identification of protein-metabolite interactions in complex metabolite mixtures by ligand-detected NMR spectroscopy.
Biochemistry. 2016 Apr 11;
Authors: Nikolaev YV, Kochanowski K, Link H, Sauer U, Allain FH
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 non-covalent nature of the binding between proteins and metabolites has so far hampered the development of methods to systematically map 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, BSA) and identified metabolite binders in complex mixes of up to 33 metabolites. Ligand-detected NMR captured all of the reported protein-metabolite interactions, spanning full range of physiologically relevant Kds (low-?M to low-mM). 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.
PMID: 27065204 [PubMed - as supplied by publisher]
More...