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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.

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