Dataset Information

Mapping low-affinity/high-specificity peptide-protein interactions using ligand-footprinting mass spectrometry.

ABSTRACT: Short linear peptide motifs that are intracellular ligands of folded proteins are a modular, incompletely understood molecular interaction language in signaling systems. Such motifs, which frequently occur in intrinsically disordered protein regions, often bind partner proteins with modest affinity and are difficult to study with conventional structural biology methods. We developed LiF-MS (ligand-footprinting mass spectrometry), a method to map peptide binding sites on folded protein domains that allows consideration of their dynamic disorder, and used it to analyze a set of D-motif peptide-mitogen-activated protein kinase (MAPK) associations to validate the approach and define unknown binding structures. LiF-MS peptide ligands carry a short-lived, indiscriminately reactive cleavable crosslinker that marks contacts close to ligand binding sites with high specificity. Each marked amino acid provides an independent constraint for a set of directed peptide-protein docking simulations, which are analyzed by agglomerative hierarchical clustering. We found that LiF-MS provides accurate ab initio identification of ligand binding surfaces and a view of potential binding ensembles of a set of D-motif peptide-MAPK associations. Our analysis provides an MKK4-JNK1 structural model, which has thus far been crystallographically unattainable, a potential alternate binding mode for part of the NFAT4-JNK interaction, and evidence of bidirectional association of MKK4 peptide with ERK2. Overall, we find that LiF-MS is an effective noncrystallographic way to understand how short linear motifs associate with specific sites on folded protein domains at the level of individual amino acids.

SUBMITTER: Parker BW

PROVIDER: S-EPMC6800362 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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Mapping low-affinity/high-specificity peptide-protein interactions using ligand-footprinting mass spectrometry.

Parker Benjamin W BW Goncz Edward J EJ Krist David T DT Statsyuk Alexander V AV Nesvizhskii Alexey I AI Weiss Eric L EL

Proceedings of the National Academy of Sciences of the United States of America 20191002 42

Short linear peptide motifs that are intracellular ligands of folded proteins are a modular, incompletely understood molecular interaction language in signaling systems. Such motifs, which frequently occur in intrinsically disordered protein regions, often bind partner proteins with modest affinity and are difficult to study with conventional structural biology methods. We developed LiF-MS (ligand-footprinting mass spectrometry), a method to map peptide binding sites on folded protein domains th ...[more]

PMID: 31578253

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Project description:Measurements from hydroxyl radical footprinting (HRF) provide rich information about the solvent accessibility of amino acid side chains of a protein. Traditional HRF data analyses focus on comparing the difference in the modification/footprinting rate of a specific site to infer structural changes across two protein states, e.g., between a free and ligand-bound state. However, the rate information itself is not fully used for the purpose of comparing different protein sites within a protein on an absolute scale. To provide such a cross-site comparison, we present a new, to our knowledge, data analysis algorithm to convert the measured footprinting rate constant to a protection factor (PF) by taking into account the known intrinsic reactivity of amino acid side chain. To examine the extent to which PFs can be used for structural interpretation, this PF analysis is applied to three model systems where radiolytic footprinting data are reported in the literature. By visualizing structures colored with the PF values for individual peptides, a rational view of the structural features of various protein sites regarding their solvent accessibility is revealed, where high-PF regions are buried and low-PF regions are more exposed to the solvent. Furthermore, a detailed analysis correlating solvent accessibility and local structural contacts for gelsolin shows a statistically significant agreement between PF values and various structure measures, demonstrating that the PFs derived from this PF analysis readily explain fundamental HRF rate measurements. We also tested this PF analysis on alternative, chemical-based HRF data, showing improved correlations of structural properties of a model protein barstar compared to examining HRF rate data alone. Together, this PF analysis not only permits a novel, to our knowledge, approach of mapping protein structures by using footprinting data, but also elevates the use of HRF measurements from a qualitative, cross-state comparison to a quantitative, cross-site assessment of protein structures in the context of individual conformational states of interest.

Dataset Information

Mapping low-affinity/high-specificity peptide-protein interactions using ligand-footprinting mass spectrometry.

Publications

Mapping low-affinity/high-specificity peptide-protein interactions using ligand-footprinting mass spectrometry.

Similar Datasets

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