Capturing Protein–Protein Interactions with Acidic Amino Acids Reactive Cross‐Linkers

Acidic residues (Asp and Glu) have a high prevalence on protein surfaces, but cross-linking reactions targeting these residues are limited. Existing methods either require high-concentration coupling reagents or have low structural compatibility. Here a previously reported "plant-and-cast" strategy is extended to develop heterobifunctional cross-linkers. These cross-linkers first react rapidly with Lys sidechains and then react with Asp and Glu sidechains, in a proximity-enhanced fashion. The cross-linking reaction proceeds at neutral pH and room temperature without coupling reagents. The efficiency and robustness of cross-linking using model proteins, ranging from small monomeric proteins to large protein complexes are demonstrated. Importantly, it is shown that this type of cross-linkers are efficient at identifying protein-protein interactions involving acidic domains. The Cross-linking mass spectrometry (XL-MS) study with p53 identified 87 putative binders of the C-terminal domain of p53. Among them, SARNP, ZRAB2, and WBP11 are shown to regulate the expression and alternative splicing of p53 target genes. Thus, these carboxylate-reactive cross-linkers will further expand the power of XL-MS in the analysis of protein structures and protein-protein interactions.