Functional Analysis of γ-Tubulin Complex Proteins Indicates Specific Lateral Association via Their N-terminal Domains.

Microtubules are nucleated from multiprotein complexes containing γ-tubulin and associated γ-tubulin complex proteins (GCPs). Small complexes (γTuSCs) comprise two molecules of γ-tubulin bound to the C-terminal domains of GCP2 and GCP3. γTuSCs associate laterally into helical structures, providing a structural template for microtubule nucleation. In most eukaryotes γTuSCs associate with additional GCPs (4, 5, and 6) to form the core of the so-called γ-tubulin ring complex (γTuRC). GCPs 2-6 constitute a family of homologous proteins. Previous structural analysis and modeling of GCPs suggest that all family members can potentially integrate into the helical structure. Here we provide experimental evidence for this model. Using chimeric proteins in which the N- and C-terminal domains of different GCPs are swapped, we show that the N-terminal domains define the functional identity of GCPs, whereas the C-terminal domains are exchangeable. FLIM-FRET experiments indicate that GCP4 and GCP5 associate laterally within the complex, and their interaction is mediated by their N-terminal domains as previously shown for γTuSCs. Our results suggest that all GCPs are incorporated into the helix via lateral interactions between their N-terminal domains, whereas the C-terminal domains mediate longitudinal interactions with γ-tubulin. Moreover, we show that binding to γ-tubulin is not essential for integrating into the helical complex.

Virtual and biophysical screening targeting the γ-tubulin complex--a new target for the inhibition of microtubule nucleation.

Microtubules are the main constituents of mitotic spindles. They are nucleated in large amounts during spindle assembly, from multiprotein complexes containing γ-tubulin and associated γ-tubulin complex proteins (GCPs). With the aim of developing anti-cancer drugs targeting these nucleating complexes, we analyzed the interface between GCP4 and γ-tubulin proteins usually located in a multiprotein complex named γ-TuRC (γ-Tubulin Ring Complex). 10 ns molecular dynamics simulations were performed on the heterodimers to obtain a stable complex in silico and to analyze the residues involved in persistent protein-protein contacts, responsible for the stability of the complex. We demonstrated in silico the existence of a binding pocket at the interface between the two proteins upon complex formation. By combining virtual screening using a fragment-based approach and biophysical screening, we found several small molecules that bind specifically to this pocket. Sub-millimolar fragments have been experimentally characterized on recombinant proteins using differential scanning fluorimetry (DSF) for validation of these compounds as inhibitors. These results open a new avenue for drug development against microtubule-nucleating γ-tubulin complexes.

Assembly of gamma-tubulin ring complexes: implications for cell biology and disease.

Microtubules are among the main constituents of the cytoskeleton. They are assembled from dimers of alpha- and beta-tubulin. This assembly occurs preferentially at organizing centers such as the centrosomes, catalyzed by multiprotein complexes of gamma-tubulin. At the beginning of mitosis, the amount of gamma-tubulin complexes at the centrosomes increases sharply, supporting the sudden formation of numerous spindle microtubules. Recent studies on the structure of gamma-tubulin complex proteins have advanced our understanding of the assembly process of gamma-tubulin complexes, and have pointed toward putative mechanisms of microtubule nucleation. Moreover, the discovery of novel proteins associated with gamma-tubulin complexes has illustrated the possibilities of how gamma-tubulin might be recruited and regulated at specific sites of microtubule organization. This chapter highlights recent developments in the field and discusses the potential of the gamma-tubulin complex as a pharmacological target, to control proliferation of cells.

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

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.

Spindle assembly defects leading to the formation of a monopolar mitotic apparatus.

Mitotic spindle formation in animal cells involves microtubule nucleation from two centrosomes that are positioned at opposite sides of the nucleus. Microtubules are captured by the kinetochores and stabilized. In addition, microtubules can be nucleated independently of the centrosome and stabilized by a gradient of Ran-GTP, surrounding the mitotic chromatin. Complex regulation ensures the formation of a bipolar apparatus, involving motor proteins and controlled polymerization and depolymerization of microtubule ends. The bipolar apparatus is, in turn, responsible for faithful chromosome segregation. During recent years, a variety of experiments has indicated that defects in specific motor proteins, centrosome proteins, kinases and other proteins can induce the assembly of aberrant spindles with a monopolar morphology or with poorly separated poles. Induction of monopolar spindles may be a useful strategy for cancer therapy, since ensuing aberrant mitotic exit will usually lead to cell death. In this review, we will discuss the various underlying molecular mechanisms that may be responsible for monopolar spindle formation.

NEDD1-dependent recruitment of the gamma-tubulin ring complex to the centrosome is necessary for centriole duplication and spindle assembly.

The centrosome is the major microtubule organizing structure in somatic cells. Centrosomal microtubule nucleation depends on the protein gamma-tubulin. In mammals, gamma-tubulin associates with additional proteins into a large complex, the gamma-tubulin ring complex (gammaTuRC). We characterize NEDD1, a centrosomal protein that associates with gammaTuRCs. We show that the majority of gammaTuRCs assemble even after NEDD1 depletion but require NEDD1 for centrosomal targeting. In contrast, NEDD1 can target to the centrosome in the absence of gamma-tubulin. NEDD1-depleted cells show defects in centrosomal microtubule nucleation and form aberrant mitotic spindles with poorly separated poles. Similar spindle defects are obtained by overexpression of a fusion protein of GFP tagged to the carboxy-terminal half of NEDD1, which mediates binding to gammaTuRCs. Further, we show that depletion of NEDD1 inhibits centriole duplication, as does depletion of gamma-tubulin. Our data suggest that centriole duplication requires NEDD1-dependent recruitment of gamma-tubulin to the centrosome.

NMR study of the interaction between Zn(II) ligated bleomycin and Streptoalloteichus hindustanus bleomycin resistance protein.

Bleomycin (Bm), a 1.4 kDa glycopeptide excreted by Streptomyces verticillus, is a natural antibacterial compound used in therapy as antineoplastic drug. To counteract its biological activity, cells have developed several resistance mechanisms, one of these based on proteins able to tightly bind Bm. In this paper, the interaction of Zn(2+)-Bm with the Streptoalloteichus hindustanus Bm resistance protein (ShBle) has been investigated by solution state NMR. Sequential nOe and chemical shift index have shown that the fold of the protein (in absence or presence of Bm) is identical to the previously published X-ray structure. The dimeric nature of ShBle is confirmed by the diffusion tensor as determined by NMR relaxation data. Using isotope filtered nOe experiment, intermolecular nOes between Bm and ShBle have been observed as used for modeling. While the interaction of the Bm metal binding site with ShBle appears to be uniquely defined, several conformations of the bithiazole moieties are compatible with the NMR data. Binding of Bm also induces changes of the local dynamics (stretch N85-G91), as shown by (15)N relaxation data. These results are discussed in the context of several Bm analogues able to interact with ShBle and of the recently published X-rays structures.

Penicillin-binding proteins 1a and 1b form independent dimers in Escherichia coli.

We report here that PBP1a can dimerize but does not interact with PBP1b to form PBP1a/PBP1b heterodimers in Escherichia coli. These findings support the idea of a relevant involvement of dimerization of both PBP1a and PBP1b during murein synthesis and suggest the existence of different peptidoglycan synthesis complexes.