Structure of the MKLP2 CPC-binding region. (A) A ClustalX alignment of MKLP2 sequences from different species highlighting different conserved features. (B) Crystal structure of dimeric MKLP2^596-668 at 1.43 Å shows the disulfide bond occurring between C607 of chains A and B. Electrostatic potential on the two sides of MKLP2 indicates a highly negatively charged region (red). (C) Electrostatic potential on the surface of MKLP2 fragment 596–668 shows the negatively charged (red) 636–652 region, involved in CPC interaction. (D) Western blot of siControl and siMKLP2 cells confirmed depletion of MKLP2 in untransfected control cells (vertical dash) and following rescue with GFP-MKLP2 (WT and mutants). Tubulin was used as a loading control. siControl, nontargeting siRNA. (E) SEC-MALS–calculated weight-average molar masses for MKLP2^557-668 (yellow), CPI80 (orange), and MKLP2:CPI80 (blue) are plotted versus the elution volume. Data were analyzed using the ASTRA software package.