The methyl 13 C-edited/ 13 C-filtered transferred NOE for studying protein interactions with short linear motifs
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ARTICLE
The methyl 13C‑edited/13C‑filtered transferred NOE for studying protein interactions with short linear motifs Suresh Kumar1 · Sabine R. Akabayov1 · Naama Kessler1 · Leah S. Cohen2,3 · Jacob Solanki2,3 · Fred Naider2,3 · Lewis E. Kay4,5,6,7 · Jacob Anglister1 Received: 19 March 2020 / Accepted: 12 August 2020 © Springer Nature B.V. 2020
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. Keywords Transferred NOE · Intermolecular interactions · Short linear recognition motif SLiM · p38α · MAP kinases
Introduction
Suresh Kumar and Sabine R. Akabayov have contributed equally to the work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10858-020-00340-y) contains supplementary material, which is available to authorized users.
Protein interactions with their target molecules are ubiquitous in biology and drive almost all biological processes including signaling, regulation and immune responses. There is a growing body of data, which indicates that intrinsically disordered short linear motifs (SLiMs) of proteins serve as recognition motifs for other proteins (Van Roey et al. 2014; Davey et al. 2015; Dodson et al. 2015). Indeed,
* Sabine R. Akabayov sabine‑[email protected]
4
Department of Molecular Genetics, The University of Toronto, Toronto, ON M5S1A8, Canada
* Jacob Anglister [email protected]
5
Department of Biochemistry, The University of Toronto, Toronto, ON M5S1A8, Canada
6
Department of Chemistry, The University of Toronto, Toronto, ON M5S1A8, Canada
7
Hospital for Sick Children, Program in Molecular Medicine, 555 University Avenue, Toronto, ON M5G 1X8, Canada
1
Department of Structural Biology, Weizma
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