Figure 2. The NEDD1 pinwheel associates... | Rockefeller University Press

Figure 2.

$The NEDD1 pinwheel associates with the γ-TuRC through multiple interfaces. (A) Cartoon representation of a model of the NEDD1 C-terminal tetramer. Locations of conserved F603 and F622 residues are highlighted by dashed circles. (B) Cross-section views of the NEDD1 tetramer AlphaFold model showing the predicted packing of F603 (left) and F622 (right). (C) Cartoon representation of a model of the NEDD1 pinwheel, color as in Fig. 1 F. Black circles indicate zoom in areas of interest for panels D and F. (D) Zoom in view of NEDD1 pinwheel AlphaFold model for regions specified in C, showing conserved residues involved in electrostatic interactions between NEDD1 and GCP3 in the pinwheel. (E) Western blot of inputs and bound fractions of SBP pulldowns of Myc-SBP-NEDD1 constructs from HEK293T cells. Untransfected cells served as a negative control. (F) Zoom in view of the fishtail region of the NEDD1 pinwheel model, highlighting previously reported mutations that abolish NEDD1:γ-TuRC interactions (maroon) (Manning et al., 2010), as well as an identified Plk1 phosphorylation site (yellow) (Zhang et al., 2009). (G) Western blot of inputs and bound fractions of SBP pulldowns of Myc-SBP-NEDD1 constructs from HEK293T cells. 1–634 refers to a NEDD1 fishtail deletion lacking residues 635–660. Untransfected cells served as a negative control. (H) Two views of the NEDD1 pinwheel Blades A and B bound to the base of the γ-TuRC (cartoon representation with cryo-EM density in transparent grey surface). (I) Lumenal view of an AlphaFold prediction of the NEDD1 pinwheel contacting the GRIP1 domains of GCP4–6. The model on the right is colored according to the pLDDT. pLDDT, predicted local distance difference test. Experiments in E and G were performed three times with similar results. Source data are available for this figure: SourceData F2.$

The NEDD1 pinwheel associates with the γ-TuRC through multiple interfaces. (A) Cartoon representation of a model of the NEDD1 C-terminal tetramer. Locations of conserved F603 and F622 residues are highlighted by dashed circles. (B) Cross-section views of the NEDD1 tetramer AlphaFold model showing the predicted packing of F603 (left) and F622 (right). (C) Cartoon representation of a model of the NEDD1 pinwheel, color as in Fig. 1 F. Black circles indicate zoom in areas of interest for panels D and F. (D) Zoom in view of NEDD1 pinwheel AlphaFold model for regions specified in C, showing conserved residues involved in electrostatic interactions between NEDD1 and GCP3 in the pinwheel. (E) Western blot of inputs and bound fractions of SBP pulldowns of Myc-SBP-NEDD1 constructs from HEK293T cells. Untransfected cells served as a negative control. (F) Zoom in view of the fishtail region of the NEDD1 pinwheel model, highlighting previously reported mutations that abolish NEDD1:γ-TuRC interactions (maroon) (Manning et al., 2010), as well as an identified Plk1 phosphorylation site (yellow) (Zhang et al., 2009). (G) Western blot of inputs and bound fractions of SBP pulldowns of Myc-SBP-NEDD1 constructs from HEK293T cells. 1–634 refers to a NEDD1 fishtail deletion lacking residues 635–660. Untransfected cells served as a negative control. (H) Two views of the NEDD1 pinwheel Blades A and B bound to the base of the γ-TuRC (cartoon representation with cryo-EM density in transparent grey surface). (I) Lumenal view of an AlphaFold prediction of the NEDD1 pinwheel contacting the GRIP1 domains of GCP4–6. The model on the right is colored according to the pLDDT. pLDDT, predicted local distance difference test. Experiments in E and G were performed three times with similar results. Source data are available for this figure: SourceData F2.

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