Asymmetric Molecular Architecture of the Human γ-Tubulin Ring Complex

Abstract

The γ-tubulin ring complex (γ-TuRC) is an essential regulator of centrosomal and acentrosomal microtubule formation, yet its structure is not known. Here, we present a cryo-EM reconstruction of the native human γ-TuRC at ∼3.8 Å resolution, revealing an asymmetric, cone-shaped structure. Pseudo-atomic models indicate that GCP4, GCP5, and GCP6 form distinct Y-shaped assemblies that structurally mimic GCP2/GCP3 subcomplexes distal to the γ-TuRC "seam." We also identify an unanticipated structural bridge that includes an actin-like protein and spans the γ-TuRC lumen. Despite its asymmetric architecture, the γ-TuRC arranges γ-tubulins into a helical geometry poised to nucleate microtubules. Diversity in the γ-TuRC subunits introduces large (>100,000 Ų) surfaces in the complex that allow for interactions with different regulatory factors. The observed compositional complexity of the γ-TuRC could self-regulate its assembly into a cone-shaped structure to control microtubule formation across diverse contexts, e.g., within biological condensates or alongside existing filaments.

Keywords: GCP2; GCP3; GCP4; GCP5; GCP6; actin; microtubule nucleation; microtubules; single particle cryo-EM; γ-tubulin ring complex.

Figure 1.. Cryo-EM reconstruction of the native human γ-TuRC.

A) Two views of the overall γ-TuRC density map (surface representation). Structural features and dimensions of the γ-TuRC are indicated. Schematic of the γ-TuRC highlighting a Y-shaped subcomplex (indicated) is shown in the bottom right. B) and C) Schematics of the γ-TuRC highlighting proposed subunit numbering, features of asymmetry (both compositional and structural), and the “overlap” region, viewed from the top (B) and side (C). D) and E) de novo molecular model for the γ-TuRC (cartoon representation), viewed from the top (D) and side (E). F) Refined γ-tubulin model (cartoon representation) with guanine nucleotide (stick representation) and helix H1 indicated. G) Examples of γ-tubulin density quality. Refined models for GDP (left, stick representation) and helix H1 of γ-tubulin (right, stick representation) shown in the corresponding γ-tubulin density (blue mesh). H) View of two γ-tubulin models at positions 8 (light blue cartoon representation) and 7 (blue cartoon representation), highlighting the γ-tubulin:γ-tubulin interface (dashed rectangle). I) Inset from H) showing interacting helices (labeled cylinders). Except for H) and I), γ-TuRC subunits are colored according to the legend in A). See also Figures S1-S3 and Tables S1-S5.

Figure 2.. Positioning of GCP4 and GCP5 in the γ-TuRC.

A) Schematic of the γ-TuRC establishing the viewing angle and highlighting the locations of GCP4 (yellow) and GCP5 (orange). B) Schematic of the γ-tubulin/GCP4/GCP5 heterotetramer that forms a Y-shaped γ-TuSC-like assembly, γ-TuG4/5. C) The density map of γ-TuRC positions 9-11 used to model N- and C-domains and short C-domain hairpins of GCP4 (yellow surface) and GCP5 (orange surface), viewed from the angle indicated in A). D) and E) Molecular models (left) of GCP4 and GCP5. Insets (right) show the fit of a region of the models in the density maps (mesh). See also Figures S2-S4 and Tables S1, S4 & S5.

Figure 3.. Positioning of GCP4 and GCP6 in the γ-TuRC.

A) Schematic of the γ-TuRC highlighting the locations of GCP4 (yellow) & GCP6 (red). B) Schematic of a γ-tubulin/GCP4/GCP6 heterotetramer that forms the γ-TuSC-like assembly, γ-TuG4/6 and interacting coiled-coil (CC) and helical bundle (HB) of the unassigned densities (dark gray). C) Density at positions 11-13 used to model N- and C-domains and short C-domain hairpins (indicated) of the second GCP4 (yellow surface) and GCP6 (red surface), viewed from the angle indicated in A). CC and HB densities interacting with position 13 are indicated. D) Molecular model of GCP4 at position 11 (left). Inset (right) shows the fit of GCP4 residues Y85-G113 (indicated; stick representation) in the density map (mesh). E) Molecular model of GCP6 at position 12 (left). Poly-alanine models of the CC and HB densities are shown for consistency with the observed density (indicated). Inset (right) shows the fit of GCP6 residues L456-A474 (indicated; stick representation) in the density map (mesh). See also Figures S2 & S4 and Tables S1, S2, S4 & S5.

Figure 4.. Structures and arrangement of human GCP2 and GCP3.

A) Schematic of the γ-TuRC highlighting locations of GCP2 (purple) and GCP3 (pink). B) Schematic of a γ-tubulin/GCP2/GCP3 heterotetramer that forms the human γ-TuSC. C) Overall density map used to model GCP2 (position 7, purple surface), GCP3 (position 8, pink surface), and the staple (grey surface) viewed from the angle indicated in A). N- and C-domains, long (GCP3) and short (GCP2) C-domain hairpins, and the staple density, are indicated. D) Molecular model of GCP2 (left). Inset shows the fit of GCP2 residues A319-Q344 (stick representation) in the density map (mesh). E) Molecular model of GCP3 (left). Inset shows the fit of GCP3 residues Q322-H343 (stick representation) in the density map (mesh). F) Poly-alanine model of the staple (cartoon representation). Secondary structure features are indicated. G) Molecular model of the human γ-TuSC comprising GCP2, GCP3, and two copies of γ-tubulin. The staple is also shown to be consistent with the density map in C). H) Rotated GCP2 (purple surface) and GCP3 (pink surface) models highlighting the intra-γ-TuSC interface (blue surface). See also Figures S2 & S5 and Tables S1, S4 & S5.

Figure 5.. Composition of the γ-TuRC “overlap” and lumenal bridge regions.

A) Side view of the γ-TuRC cryo-EM density map highlighting subunits leading up to the γ-TuRC “overlap” (indicated). A density at position 14 (asterisk) extending into the “overlap” region (dashed line) from an arrangement of GCP2 & the tentatively-assigned GCP3 (the “terminal γ-TuSC”) is indicated. B) Schematic of the γ-TuSC at the overlap adjacent to γ-TuG4/6 indicating its ~6° rotation relative to a hinge point in the γ-tubulin at position 13 (green star). The GCP6 “plug” is omitted for clarity. C) Top view of the overall γ-TuRC density map highlighting the lumenal bridge (dashed rectangle, spanning from positions 2 and 3 to positions 9 and 10) and its two sub-domains (i: bi-lobed density domain; ii: α-helical bundle domain). D) Zoomed-in view of the α-helical bundle domain ((ii) in C); grey surface) that interacts (dashed circle) with density (white surface) stemming from GCP5 (orange surface). E) Left: Zoomed-in view of the interactions (white dashed circles) between the bi-lobed density ((i) in C); grey surface) and γ-tubulins at positions 2 and 3 (blue surface). Right: A rigid-body fit of β-actin (grey cartoon representation; PDB ID 2HF3; (Rould et al., 2006)) in the bi-lobed density (transparent grey surface). See also Figures S5-S6 and Tables S1, S2, S4 & S5.

Figure 6.. Asymmetric molecular architecture of the γ-TuRC.

A) Schematic of γ-TuRC organization. The γ-TuRC consists of two highly asymmetric, but similarly-sized halves: 1) a γ-TuSC oligomer-like “core” (salmon arrow); and 2) an arrangement of divergent γ-TuSC-like subunits (γ-TuG4/5 & γ-TuG4/6) capped by a terminal γ-TuSC that, together, form a large and diverse binding surface for regulatory factors (green arc). The γ-tubulins are not shown for clarity. B) Left: Two views of the quasi-helical arrangement of γ-tubulin models in the human γ-TuRC. Right: Two views of the helical arrangement of α- (grey) and β- (white) tubulin dimers within the microtubule lattice (PDB: 6E7B; Ti et al., 2018). The helical rise (indicated) and diameter of the γ-tubulin ring do not perfectly match the microtubule lattice.