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

Abstract 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.