Mitotic spindle association of TACC3 requires Aurora‐A‐dependent stabilization of a cryptic α‐helix

Aurora‐A regulates the recruitment of TACC3 to the mitotic spindle through a phospho‐dependent interaction with clathrin heavy chain (CHC). Here, we describe the structural basis of these interactions, mediated by three motifs in a disordered region of TACC3. A hydrophobic docking motif binds to a previously uncharacterized pocket on Aurora‐A that is blocked in most kinases. Abrogation of the docking motif causes a delay in late mitosis, consistent with the cellular distribution of Aurora‐A complexes. Phosphorylation of Ser558 engages a conformational switch in a second motif from a disordered state, needed to bind the kinase active site, into a helical conformation. The helix extends into a third, adjacent motif that is recognized by a helical‐repeat region of CHC, not a recognized phospho‐reader domain. This potentially widespread mechanism of phospho‐recognition provides greater flexibility to tune the molecular details of the interaction than canonical recognition motifs that are dominated by phosphate binding. Synopsis Clathrin‐mediated TACC3 recruitment to mitotic spindles depends on Aurora A phosphorylation. This is now shown to involve phosphorylation‐mediated conformational changes rather than direct phosphate group recognition, exemplifying a flexible alternative mechanism of phospho‐regulation. Two crystal structures reveal snapshots of the mitotic TACC3/clathrin complex and its regulation by Aurora‐A. TACC3 docks to a selective site on the N‐lobe of Aurora‐A via a hydrophobic sequence. Phosphorylation of Ser558 switches the local conformation of TACC3 from disordered to helical. The cryptic TACC3 helix is recognized by a clathrin region lacking canonical phospho‐reader motifs. Graphical Abstract Regulated TACC3 recruitment by clathrin involves phosphorylation‐mediated conformational changes rather than direct phosphate group recognition, exemplifying a flexible alternative mechanism of phospho‐regulation.