Among those it recruits and phosphorylates is Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3). TACC3 is instrumental for spindle assembly and chromosome segregation in mammalian cells, playing a central role in achieving microtubule stabilization during mitosis through its interaction with chTOG/XMAP215. 26,27 TACC3 is mutated and overexpressed in different cancer types, including glioblastoma, with its fusion products also exerting oncogenic activity. Moreover, depletion or pharmacological inhibition of Aurora-A kinase activity by small-molecule inhibitors has been shown to disrupt centrosomal localization of TACC3, and suppress tumor growth, whilst siRNA-mediated TACC3 depletion leads to a similar phenotype to that which occurs through Aurora-A active-site inhibition. Burgess et al. reported an x-ray crystal structure for a fragment of TACC3 (residues 519-563) bound to Aurora-A122-403 C290A/C393A/D274N (PDB: 5ODT; Fig. ). The TACC3 fragment was shown to adopt an extended conformation with two short regions adopting secondary structure: a 310-helical turn (residues 527-531), and an α-helix (residues 546-555, the αTR domain, Fig. ). Fluorescence anisotropy (FA) titrations established that TACC3522-536encompassing the 310-helical turn -makes the dominant contribution to Aurora-A affinity (~10-20 µM). Microscale thermophoresis further identified Phe525 TACC3 as a hot-spot residue for Aurora-A binding. Moreover, HeLa cells that express a F525A variant of TACC3 generated using CRISPR-Cas9 exhibited a phenotype consistent with critical roles of this hotspot in early and late mitosis. Our first objective was to optimize the affinity of TACC3522-536 for Aurora-A. Our analyses of the TACC3/Aurora-A co-crystal structure identified three interactions that might play a role in influencing the affinity of TACC3 for Aurora-A: a charge-reinforced hydrogen-bond between Glu523 TACC3 and Arg137 Aurora-A ; a hydrophobic contact between Leu532 TACC3 and Tyr148 Aurora-A ; and, a cation-π interaction between Phe525 TACC3 and Arg151 Aurora-A . These analyses were supported through in silico alanine scanning (Table ).