Proximity-enhanced cysteine–histidine crosslinking for elucidating intrinsically disordered and other protein complexes

Disordered proteins and domains are ubiquitous throughout the proteome of human cell types, yet the biomolecular sciences lack effective tool compounds and chemical strategies to study this class of proteins. In this context, we introduce a novel covalent tool compound approach that combines proximity-enhanced crosslinking with histidine trapping. Utilizing a maleimide-cyclohexenone crosslinker for efficient cysteine-histidine crosslinking, we elucidated the mechanism of this dual-reactive tool compound class. This tool compound concept was then applied to profile the full-length complex of 14-3-3 and hyperphosphorylated Tau (hpTau), relevant to Alzheimer's. This approach identified a cryptic binding interaction between 14-3-3 and hpTau <i>via</i> its phosphorylated Ser356, overlooked by the majority of 14-3-3/Tau literature. Utilizing a mutational study and an equilibrium model, this cryptic binding interaction is revealed to play a prominent biomolecular role at cellularly relevant concentrations. This finding necessitates a re-evaluation of the mechanism of the 14-3-3/Tau interaction. The histidine-trap crosslinker approach reported here not only advances our understanding of the 14-3-3/Tau interaction but also demonstrates the potential of dual-covalent tool compounds in studying complex interactions involving IDPs and IDDs.