Abstract
Many proteins interact with their ligand proteins by recognition of short linear motifs that are often intrinsically disordered. These interactions are usually weak and are characterized by fast exchange. NMR spectroscopy is a powerful tool to study weak interactions. The methods that have been commonly used are analysis of chemicals shift perturbations (CSP) upon ligand binding and saturation transfer difference spectroscopy. These two methods identify residues at the binding interface between the protein and its ligand. In the present study, we used a combination of transferred-NOE, specific methyl-labeling and an optimized isotope-edited/isotope-filtered NOESY experiment to study specific interactions between the 42 kDa p38α mitogen-activated protein kinase and the kinase interaction motif (KIM) on the STEP phosphatase. These measurements distinguished between residues that both exhibit CSPs upon ligand binding and interact with the KIM peptide from residues that exhibit CSPs but do not interact with the peptide. In addition, these results provide information about pairwise interactions that is important for a more reliable docking of the KIM peptide into its interacting surface on p38α. This combination of techniques should be applicable for many protein-peptide complexes up to 80 kDa for which methyl resonance assignment can be achieved.
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Acknowledgements
We thank Dr. Tali Scherf for help in setting up some of the NMR experiments. This study was supported by The Israel Academy of Sciences, The US-Israel Binational Science Foundation and by the Kimmelman Center (JA), J.A. is the Dr. Joseph and Ruth Owades Professor of Chemistry.
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JA designed the experiments and wrote the paper. FN helped to write the paper and contributed essential reagents used in preliminary studies. LEK developed the pulse sequences described in the study. SK prepared the labeled proteins. SK, SA and NK analyzed the data, and helped writing the paper. LSC and JS synthesized PTP peptides used in preliminary investigations.
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Kumar, S., Akabayov, S.R., Kessler, N. et al. The methyl 13C-edited/13C-filtered transferred NOE for studying protein interactions with short linear motifs. J Biomol NMR 74, 681–693 (2020). https://doi.org/10.1007/s10858-020-00340-y
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DOI: https://doi.org/10.1007/s10858-020-00340-y