Figure 2. Modeling of mNup133 recognition by the mCenp‐F and validation by Y2H of mutants designed to impair Cenp‐F interaction with Nup133.

1. Predicted Model 3, involving interaction of mCenp‐F‐SID (parallel coiled‐coil, brown) with helix α1 (red) of mNup133 β‐propeller (green). Two orthogonal views are presented in panels (i) and (ii). In (i), Cenp‐F mutated residues impairing dimerization L2669G/L2696G are highlighted. (iii) presents a detailed view of the interface, highlighting the interaction between ^mCenp‐FR2687 and ^mNup133E93. Mutations designed to test this model are indicated as sticks and labeled in panels (i) and (iii). The colored spheres in (i) and (ii) represent the center of mass of Cenp‐F dimeric segments for the 10 most likely binding modes of Cenp‐F to Nup133 as predicted by InterEvDock server (M1 to M10) 41.
2. Y2H interactions between the indicated fragments of mNup133 and mCenp‐F (‐Ct1, ‐Ct2, or ‐SID), either WT or bearing the indicated mutations, were assayed based on growth on ‐LWH medium. The corresponding Western blots are presented in Appendix Fig S2A(ii) and B(i).