Crystal structure of γ-tubulin complex protein GCP4 provides insight into microtubule nucleation

Abstract

Microtubule nucleation in all eukaryotes involves γ-tubulin small complexes (γTuSCs) that comprise two molecules of γ-tubulin bound to γ-tubulin complex proteins (GCPs) GCP2 and GCP3. In many eukaryotes, multiple γTuSCs associate with GCP4, GCP5 and GCP6 into large γ-tubulin ring complexes (γTuRCs). Recent cryo-EM studies indicate that a scaffold similar to γTuRCs is formed by lateral association of γTuSCs, with the C-terminal regions of GCP2 and GCP3 binding γ-tubulin molecules. However, the exact role of GCPs in microtubule nucleation remains unknown. Here we report the crystal structure of human GCP4 and show that its C-terminal domain binds directly to γ-tubulin. The human GCP4 structure is the prototype for all GCPs, as it can be precisely positioned within the γTuSC envelope, revealing the nature of protein-protein interactions and conformational changes regulating nucleation activity.

GCP6 is a substrate of Plk4 and required for centriole duplication

“…Interestingly, this region is conserved down to the Xenopus laevis GCP6 orthologue Xgrip210 (Zhang et al, 2000). Whereas the grip domains were reported to be required for the interaction between GCPs and ctubulin during complex formation, the interface between the two conserved domains is assumed to form a flexible hinge in GCP3 (Guillet, 2011;Kollman et al, 2008). Conformational changes in this flexible region, which might be regulated by posttranslational modifications, were suggested to be required for triggering the microtubule-nucleating activity of the c-TuRC (Guillet, 2011;Kollman et al, 2008).…”

“…Whereas the grip domains were reported to be required for the interaction between GCPs and ctubulin during complex formation, the interface between the two conserved domains is assumed to form a flexible hinge in GCP3 (Guillet, 2011;Kollman et al, 2008). Conformational changes in this flexible region, which might be regulated by posttranslational modifications, were suggested to be required for triggering the microtubule-nucleating activity of the c-TuRC (Guillet, 2011;Kollman et al, 2008). Because the majority of Plk4 phosphorylation sites were identified in a corresponding region between the grip1 and grip2 domains of GCP6, including the repeat region, it is tempting to speculate that a phosphorylation of GCP6 by Plk4 activates centriolar microtubule nucleation and thereby procentriole formation.…”

“…One of these (Ser392) was located within the Nterminal grip1 domain (Fig. 6B), which describes a region conserved among the different GCPs (Guillet, 2011). The two other phosphorylation sites (Ser1437 and Ser1465) were located in close proximity to the C-terminal grip2 domain (Fig.…”

The where, when and how of microtubule nucleation – one ring to rule them all

“…Highly conserved sequences in GCPs 2-6 were initially described as c-tubulin ring protein (Grip) motifs (Gunawardane et al, 2000), but the sequence similarity extends beyond these motifs and, on the basis of insight obtained from the GCP4 crystal structure, we will refer to the conserved regions as N-and C-terminal 'Grip domains' (Fig. 1A) (Guillet et al, 2011). More-recent studies have identified Fig.…”

“…1A), and that GCP4, GCP5 and GCP6 might be part of the cTuRC ring structure by substituting for GCP2 or GCP3 at specific positions to function, for example, as ring assembly initiators or terminators (Fig. 1B) (Guillet et al, 2011;Kollman et al, 2011).…”