Mechanistic Design of Cell-Penetrating Disruptors for a Phospho-Dependent Interaction

The complex formed by transforming acidic coiled coil 3 (TACC3) and clathrin heavy chain (CHC) enhances mitotic spindle stability and strength by cross-linking microtubules. The interaction is dependent on phosphorylation of TACC3 at S558 by Aurora-A. Previously, we elucidated the structural basis of the TACC3/CHC interaction, which is driven by hydrophobic residues on both proteins and the formation of an α-helix in TACC3 that docks into the helical repeats of CHC. Here we find that this phosphorylation event plays an unusual role in the protein-protein interaction; rather than direct bond formation, the phosphorylated residue acts by overcoming an inherent electrostatic repulsion between K507 of CHC and basic residues in TACC3. Leveraging this insight, we optimized the sequence using peptide arrays to develop a hydrocarbon-stapled peptide (SP TACC3) that binds CHC with over a hundred-fold higher affinity than the parental TACC3 peptide, effectively disrupting the native interaction. The crystal structure of the SP TACC3/CHC complex reveals the basis for the enhanced interaction and highlights the contribution of additional polar and hydrophobic interactions. SP TACC3 efficiently penetrates cells and displaces TACC3 from the mitotic spindle, causing a delay in mitotic progression in two out of three cancer cell lines. This work showcases the novel application of hydrocarbon-stapled peptides to disrupt the TACC3/CHC protein-protein interaction in a cellular context, highlighting the potential of targeting this interface for future cancer therapies.