Figure 1.

Structure determination of the Spc24-25/Cnn1 interface. (A) Schematic representation illustrating the general centromere-microtubule orientation of the Cnn1–Ndc80 complex. Nuf2 and Ndc80 are shown in brown and dark grey, respectively, with the microtubule-binding calponin homology (CH) domains highlighted; Spc24 and Spc25 are shown in light grey and slate blue, respectively. The Cnn1 conserved N-terminal motif (65–79) is displayed in orange and its histone-fold domain (HFD) is highlighted. A rectangle indicates the interface visualized in the crystal structure. (B) Isothermal titration calorimetry performed by titrating the Spc24-25 globular domain (Spc24-25G) with a Cnn1 peptide including the Ndc80-binding motif (Cnn1^60–84). The K_d was estimated fitting a non-linear curve to the binding isotherms derived from the data. (C) Overall architecture of the Spc24^155–213–Spc25^133–221–Cnn1^60–84 crystal structure. Spc24, Spc25, and Cnn1 are represented according to their secondary structure and coloured in light grey, slate blue, and orange, respectively. Arrows indicate the opposite N-terminal/C-terminal orientation of Cnn1 relative to Spc24-25. A dotted line indicates the residue Lys184 of Spc24 not visible in the electron density (molecule B-A-F of the asymmetric unit). (D) Localization of Cnn1^60–84 in the hydrophobic pocket at the interface between Spc24^155–213 and Spc25^133–221, shown as electrostatic surface (transparency 20%). Cnn1 secondary structure is displayed in orange.