Figure 3.

Validation of the Spc24-25/Cnn1 interface. (A) Role of L160 (Spc24) and V159 (Spc25) in Cnn1^60–84 binding. The secondary structure of Spc24, Spc25, and Cnn1 is displayed in light grey, slate blue, and orange, respectively (transparency 40%). The residues L160 (Spc24) and V159 (Spc25), mutated to aspartic acid for validating the Spc24^155–213–Spc25^133–221/Cnn1^60–84 interface, are shown as sticks and highlighted in yellow. The Cnn1 residues that establish van der Waals contacts with L160 or V159 are displayed as sticks. (B) Elution profiles of size-exclusion chromatography performed with Spc24-25 globular domain (Spc24-25G) wild-type (in red) or Cnn1-binding mutants, Spc24-25G^25V159D (in violet) and Spc24-25G^24L160D (in magenta). Identical elution profiles indicate a similar complex shape and integrity. (C) Isothermal titration calorimetry performed by titrating Spc24-25G^25V159D or Spc24-25G^24L160D with Cnn1^60–84 (n.b.=no binding). (D) Coomassie-stained gels of analytical size-exclusion chromatography using 12 μM Cnn1ΔHFD (1–270), 12 μM full-length Spc24-25 wild-type and their combination (upper part); 12 μM full-length Spc25-25^V159D and its combination in equimolar amount with Cnn1ΔHFD (lower part). Complex formation is visualized by a shift to earlier elution volumes of both molecules.