Structure of the microtubule anchoring factor NEDD1 bound to the γ-tubulin ring complex

Abstract

The γ-tubulin ring complex (γ-TuRC) is an essential multiprotein assembly, in which γ-tubulin, GCP2-6, actin, MZT1 and MZT2 form an asymmetric cone-shaped structure that provides a template for microtubule nucleation. The γ-TuRC is recruited to microtubule organizing centers (MTOCs), such as centrosomes and pre-existing mitotic spindle microtubules, via the evolutionarily-conserved attachment factor NEDD1. NEDD1 contains an N-terminal WD40 domain that binds to microtubules, and a C-terminal domain that associates with the γ-TuRC. However, the structural basis of the NEDD1-γ-TuRC interaction is not known. Here, we report cryo-electron microscopy (cryo-EM) structures of NEDD1 bound to the human γ-TuRC in the absence or presence of the activating factor CDK5RAP2, which interacts with GCP2 to induce conformational changes in the γ-TuRC and promote its microtubule nucleating function. We found that the C-terminus of NEDD1 forms a tetrameric α-helical assembly that contacts the lumen of the γ-TuRC cone, is anchored to GCP4, 5 and 6 via protein modules consisting of MZT1 & GCP3 subcomplexes, and orients its microtubule-binding WD40 domains away from the complex. We biochemically tested our structural models by identifying NEDD1 mutants unable to pull-down γ-tubulin from cultured cells. The structure of the γ-TuRC simultaneously bound to NEDD1 and CDK5RAP2 reveals that both factors can associate with the "open" conformation of the complex. Our results show that NEDD1 does not induce conformational changes in the γ-TuRC, but suggest that anchoring of γ-TuRC-capped microtubules by NEDD1 would be structurally compatible with the significant conformational changes experienced by the γ-TuRC during microtubule nucleation.

Figure 1.. A pinwheel-shaped structure consisting of a tetrameric NEDD1 helical bundle and four MZT1:GCP3-NHD modules docks onto the base of the asymmetric cone-shaped human γ-TuRC.

A) Schematic of the human NEDD1 sequence. A zoom-in on the C-terminal helical region predicted to form several α-helices is shown. Secondary structure predictions are taken from UniProt (UniProt Consortium, 2023). B) Cartoon representation of the MZT1:GCP3-NHD structure in the γ-TuRC lumenal bridge, from PDB ID: 6X0U (Wieczorek et al., 2020a). C) Two views of an AlphaFold prediction containing 4 copies each of NEDD1 residues 571–660, MZT1, and GCP3 residues 1–120. The model on the left is coloured according to the predicted local distance difference test (pLDDT) score from the AlphaFold prediction. D) Two views of the consensus recombinant γ-TuRC density map (surface representation). The seam, lumenal bridge, and pinwheel-shaped densities are labeled; the pinwheel-shaped density is coloured in lavender. Map resolution is 4.7 Å, but is shown at a low threshold to include features with weaker density. Higher resolution features can be found in Supplementary Figure S3B. E) Two views of the pinwheel density post-processed using EMready (He et al., 2023) (light pink surface representation). The CryoSPARC post-processed map is shown at the same threshold as in D) as a transparent white surface for reference. The pinwheel axle containing the fish tail and α-helical tetramer, as well as pinwheel blades A-D, are indicated. F) Two views of the refined γ-TuRC model in the rec-γ-TuRC consensus map, focusing on the pinwheel density. Pinwheel features are labeled as in E).

Figure 2.. The NEDD1 pinwheel associates with the γ-TuRC through conserved 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. Black circles indicate zoom-in areas of interest for panels D) and E), color legend as in Figure 1F. 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. Untransfected cells served as a negative control. The experiment was performed three times with similar results. F) Zoom-in view of the fish-tail 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) 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).

Figure 3.. The NEDD1 pinwheel contacts an extension in the GCP6 belt and enables the assignment of MZT1:GCP5-NHD to the γ-TuRC seam.

A) Two views of the upper helical pair of the NEDD1 fish tail contacting positions 1 and 2 of the γ-TuRC (top: cartoon representation and cryo-EM density in transparent grey surface; middle: cartoon representation of NEDD1 and GCP6 next to a surface representation of the γ-TuRC). Newly-modeled GCP6 residues 191–252 extending from the GCP6 belt are indicated. The bottom panel shows a rotated view of the same interface to highlight the unattached helical pair in the fish tail. γ-TuRC subunit positions 1 (GCP2) and 2 (GCP3) are indicated, where possible. B) AlphaFold model of GCP3, MZT1:GCP5-NHD, and γ-tubulin. The model on the left is coloured according to the predicted local distance difference test (pLDDT) score from the AlphaFold prediction (cartoon representation). The model on the right is shown in surface representation and is coloured according to the legend. C) AlphaFold model of GCP3, MZT1:GCP3-NHD, and γ-tubulin. The model on the left is coloured according to the predicted local distance difference test (pLDDT) score from the AlphaFold prediction (cartoon representation). The model on the right is shown in surface representation and is coloured according to the legend. D) Surface representation of rigid body-fitted AlphaFold model of GCP5, GCP4, GCP6, GCP2, GCP3, and MZT1 in the rec-γ-TuRC consensus map (transparent representation). MZT1:GCP5-NHD, GCP3, and the disordered GCP5 linker (a.a. 120–200) are indicated. The Euclidean distance between GCP5 residue 120 and 200 in the model is indicated. E) Secondary structure prediction of human GCP5 residues 120–200. Top: primary sequence (red = predicted α-helices); middle: jnetpred secondary structure prediction result (red = α-helices); bottom: confidence score for the prediction. Figure generated using Jalview (Waterhouse et al., 2009).

Figure 4.. Both CDK5RAP2 and NEDD1 can simultaneously associate with the open γ-TuRC conformation.

A) Top: two views of the consensus rec-γ-TuRC + CDK5RAP2 density map (surface representation). Unassigned, NEDD1 pinwheel and CMG module densities are indicated. Map resolution is 5.1 Å but is shown at a low threshold to include features with weaker density. Bottom left: A schematic top view of the γ-TuRC’s subunit organization. Bottom right: a refined molecular model of the rec-γ-TuRC + CDK5RAP2 (cartoon representation), with zoomed-in views for the CMG module and NEDD1 pinwheel in the density (transparent surface). B) Two cartoon representation views of superimposed rec-γ-TuRC (magenta) and rec-γ-TuRC + CDK5RAP2 (blue) models. The NEDD1 pinwheel is indicated (bottom). C) Plot of Euclidean center-of-mass distances (d_COM) vs. γ-TuRC subunit position between the indicated γ-TuRC models (rec-γ-TuRC + CDK5RAP2, rec-γ-TuRC, and PDB: 6V6S as an “open” conformation control (Wieczorek et al., 2020b), all relative to PDB: 8VRK corresponding to the closed rec-γ-TuRC (Aher et al., 2024)). D) Plot of the average shift in θ vs. the shift in ϕ for helix H12 in γ-tubulins from each γ-TuRC described in (C), relative to γ-tubulins at the same positions in the closed γ-TuRC oligomer (grey circle, indicated). Standard errors in ϕ and θ are displayed as lines. The axes in (D) are scaled equally. Coloring in (D) follows the legend in (C). E) Model summarizing the findings in this study. The rec-γ-TuRC + CDK5RAP2 model has been converted to a 15 A low pass-filtered map and coloured accordingly. Unresolved WD40 domains stemming from the NEDD1 pinwheel and available to interact with binding partners are shown as hexagons. Free CMG module binding sites that should still be able to induce partial or full γ-tubulin ring closure are indicated.