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

Area of Science:

• Molecular biology
• Biochemistry
• Structural biology

Background:

• Short linear motifs are crucial intracellular ligands in cellular signaling pathways.
• These motifs, often found in disordered protein regions, exhibit moderate binding affinities and pose challenges for traditional structural studies.
• Understanding these interactions is key to deciphering complex signaling networks.

Purpose of the Study:

• To introduce and validate a novel method, ligand-footprinting mass spectrometry (LiF-MS), for mapping peptide binding sites on folded proteins.
• To analyze specific peptide-protein interactions, particularly D-motif peptide-mitogen-activated protein kinase (MAPK) associations.
• To provide structural insights into these dynamic molecular interactions.

Main Methods:

• Development of LiF-MS, employing a cleavable crosslinker to mark peptide-protein contact sites with high specificity.
• Utilizing marked amino acids as constraints for directed peptide-protein docking simulations.
• Analysis of docking simulations using agglomerative hierarchical clustering to determine binding surfaces and ensembles.

Main Results:

• LiF-MS accurately identifies ligand binding surfaces and potential binding ensembles.
• Structural models were generated for MKK4-JNK1 (previously crystallographically unattainable) and NFAT4-JNK interactions.
• Evidence for bidirectional association between MKK4 peptide and ERK2 was observed.

Conclusions:

• LiF-MS is an effective non-crystallographic technique for elucidating peptide-protein interactions at the amino acid level.
• The method provides valuable structural information for intrinsically disordered protein regions and their ligands.
• LiF-MS advances the understanding of molecular interaction languages in cellular signaling.