scholarly journals Structural convergence for tubulin binding of CPAP and vinca domain microtubule inhibitors

2021 ◽  
Author(s):  
Valérie Campanacci ◽  
Agathe Urvoas ◽  
Liza Ammar Khodja ◽  
Magali Aumont-Nicaise ◽  
Magali Noiray ◽  
...  
Keyword(s):  
Small Molecules ◽  
Binding Mode ◽  
Microtubule Assembly ◽  
Cellular Proteins ◽  
Microtubule Inhibitors ◽  
Tubulin Binding ◽  
Characteristic Features ◽  
Α Helix ◽  
Open Question ◽  
Β Tubulin

Microtubule dynamics is regulated by various cellular proteins and perturbed by small molecule compounds. To what extent the mechanism of the former resembles that of the latter is an open question. We report here structures of tubulin bound to the PN2-3 domain of CPAP, a protein controlling the length of the centrioles. We show that an α-helix of the PN2-3 N-terminal region binds and caps the longitudinal surface of the tubulin β subunit. Moreover, a PN2-3 N-terminal stretch lies in a β-tubulin site also targeted by fungal and bacterial peptide-like inhibitors of the vinca domain, sharing a very similar binding mode with these compounds. Therefore, our results identify several characteristic features of cellular partners that bind to this site and highlight a structural convergence of CPAP with small molecules inhibitors of microtubule assembly.

scholarly journals Diffusion rather than intraflagellar transport likely provides most of the tubulin required for axonemal assembly in Chlamydomonas

2020 ◽  
Vol 133 (17) ◽  
pp. jcs249805 ◽  
Author(s):  
Julie Craft Van De Weghe ◽  
J. Aaron Harris ◽  
Tomohiro Kubo ◽  
George B. Witman ◽  
Karl F. Lechtreck
Keyword(s):  
Binding Site ◽  
Critical Concentration ◽  
Intraflagellar Transport ◽  
Microtubule Assembly ◽  
Strong Reduction ◽  
Tubulin Binding ◽  
Respective Contribution ◽  
Β Tubulin

ABSTRACTTubulin enters the cilium by diffusion and motor-based intraflagellar transport (IFT). However, the respective contribution of each route in providing tubulin for axonemal assembly remains unknown. Using Chlamydomonas, we attenuated IFT-based tubulin transport of GFP–β-tubulin by altering the IFT74N–IFT81N tubulin-binding module and the C-terminal E-hook of tubulin. E-hook-deficient GFP–β-tubulin was incorporated into the axonemal microtubules, but its transport frequency by IFT was reduced by ∼90% in control cells and essentially abolished when the tubulin-binding site of IFT81 was incapacitated. Despite the strong reduction in IFT, the proportion of E-hook-deficient GFP–β-tubulin in the axoneme was only moderately reduced. In vivo imaging showed more GFP–β-tubulin particles entering cilia by diffusion than by IFT. Extrapolated to endogenous tubulin, the data indicate that diffusion provides most of the tubulin required for axonemal assembly. We propose that IFT of tubulin is nevertheless needed for ciliogenesis, because it augments the tubulin pool supplied to the ciliary tip by diffusion, thus ensuring that free tubulin there is maintained at the critical concentration for plus-end microtubule assembly during rapid ciliary growth.

scholarly journals The structure of tubulin-binding cofactor A fromLeishmania majorinfers a mode of association during the early stages of microtubule assembly

2015 ◽  
Vol 71 (5) ◽  
pp. 539-546 ◽  
Author(s):  
Keri L. Barrack ◽  
Paul K. Fyfe ◽  
William N. Hunter
Keyword(s):  
Crystal Structure ◽  
Electrostatic Interactions ◽  
Leishmania Major ◽  
Model System ◽  
Microtubule Assembly ◽  
Early Stages ◽  
Tubulin Binding ◽  
Microtubule Formation ◽  
Β Tubulin

Tubulin-binding cofactor A (TBCA) participates in microtubule formation, a key process in eukaryotic biology to create the cytoskeleton. There is little information on how TBCA might interact with β-tubulin en route to microtubule biogenesis. To address this, the protozoanLeishmania majorwas targeted as a model system. The crystal structure of TBCA and comparisons with three orthologous proteins are presented. The presence of conserved features infers that electrostatic interactions that are likely to involve the C-terminal tail of β-tubulin are key to association. This study provides a reagent and template to support further work in this area.

scholarly journals Protection from Free β-Tubulin by the β-Tubulin Binding Protein Rbl2p

2002 ◽  
Vol 22 (1) ◽  
pp. 138-147 ◽  
Author(s):  
Katharine C. Abruzzi ◽  
Adelle Smith ◽  
William Chen ◽  
Frank Solomon
Keyword(s):  
Binding Protein ◽  
In Vitro Assay ◽  
In Vitro Studies ◽  
Microtubule Assembly ◽  
Vitro Assay ◽  
Tubulin Binding ◽  
And Function ◽  
Β Tubulin ◽  
In Cells

ABSTRACT Free β-tubulin not in heterodimers with α-tubulin can be toxic, disrupting microtubule assembly and function. We are interested in the mechanisms by which cells protect themselves from free β-tubulin. This study focused specifically on the function of Rbl2p, which, like α-tubulin, can rescue cells from free β-tubulin. In vitro studies of the mammalian homolog of Rbl2p, cofactor A, have suggested that Rbl2p/cofactor A may be involved in tubulin folding. Here we show that Rbl2p becomes essential in cells containing a modest excess of β-tubulin relative to α-tubulin. However, this essential activity of Rbl2p/cofactorA does not depend upon the reactions described by the in vitro assay. Rescue of β-tubulin toxicity requires a minimal but substoichiometric ratio of Rbl2p to β-tubulin. The data suggest that Rbl2p binds transiently to free β-tubulin, which then passes into an aggregated form that is not toxic.

scholarly journals An α-Tubulin Mutant Destabilizes the Heterodimer: Phenotypic Consequences and Interactions with Tubulin-binding Proteins

1998 ◽  
Vol 9 (9) ◽  
pp. 2349-2360 ◽  
Author(s):  
Leticia R. Vega ◽  
James Fleming ◽  
Frank Solomon
Keyword(s):  
Binding Proteins ◽  
Microtubule Assembly ◽  
Wild Type ◽  
Cellular Regulation ◽  
Tubulin Binding ◽  
Cold Sensitive ◽  
Mutant Cells ◽  
Dependent Processes ◽  
Β Tubulin

Many effectors of microtubule assembly in vitro enhance the polymerization of subunits. However, several Saccharomyces cerevisiae genes that affect cellular microtubule-dependent processes appear to act at other steps in assembly and to affect polymerization only indirectly. Here we use a mutant α-tubulin to probe cellular regulation of microtubule assembly.tub1-724 mutant cells arrest at low temperature with no assembled microtubules. The results of several assays reported here demonstrate that the heterodimer formed between Tub1-724p and β-tubulin is less stable than wild-type heterodimer. The unstable heterodimer explains several conditional phenotypes conferred by the mutation. These include the lethality of tub1-724haploid cells when the β-tubulin–binding protein Rbl2p is either overexpressed or absent. It also explains why theTUB1/tub1-724 heterozygotes are cold sensitive for growth and why overexpression of Rbl2p rescues that conditional lethality. Both haploid and heterozygous tub1-724 cells are inviable when another microtubule effector, PAC2, is overexpressed. These effects are explained by the ability of Pac2p to bind α-tubulin, a complex we demonstrate directly. The results suggest that tubulin-binding proteins can participate in equilibria between the heterodimer and its components.

Crystal structure of tubulin folding cofactor A from Arabidopsis thaliana and its β-tubulin binding characterization

FEBS Letters ◽  
2010 ◽  
Vol 584 (16) ◽  
pp. 3533-3539 ◽  
Author(s):  
Lu Lu ◽  
Jie Nan ◽  
Wei Mi ◽  
Lan-Fen Li ◽  
Chun-Hong Wei ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Arabidopsis Thaliana ◽  
Tubulin Binding ◽  
Β Tubulin

scholarly journals Atomic model of microtubule-bound tau

2018 ◽  
Author(s):  
Elizabeth H. Kellogg ◽  
Nisreen M.A. Hejab ◽  
Simon Poepsel ◽  
Kenneth H. Downing ◽  
Frank DiMaio ◽  
...  
Keyword(s):  
High Resolution ◽  
Physiological Role ◽  
Biochemical Data ◽  
Disease Pathogenesis ◽  
Developmentally Regulated ◽  
Computational Approaches ◽  
Selective Stabilization ◽  
Tubulin Binding ◽  
Future Treatments ◽  
Β Tubulin

AbstractTau is a developmentally regulated protein found in axons, whose physiological role is to stabilize and bundle microtubules (MTs). Hyper-phosphorylation of tau is thought to cause its detachment from MTs and subsequent aggregation into pathological fibrils that have been implicated in Alzheimer’s disease pathogenesis. Despite its known MT binding role, there is no consensus regarding which tau residues are crucial for tau-MT interactions, where on the MT tau binds, and how binding results in MT stabilization. We have used cryo-EM to visualize the interaction of different tau constructs with MTs at high resolution (3.2-4.8 Å) and used computational approaches to generate atomic models of tau-tubulin interactions. Our work shows that the highly conserved tubulin-binding repeats within tau adopt very similar structures in their interactions with the MT. Each tau repeat binds the MT exterior and adopts an extended structure along the crest of the protofilament (PF), interacting with both α- and β-tubulin, thus stabilizing the interface between tubulin dimers. Our structures agree with and explain previous biochemical data concerning the effect of phosphorylation on MT affinity and lead to a model in which tau repeats bind in tandem along a PF, tethering together tubulin dimers and stabilizing longitudinal polymerization interfaces. These structural findings could establish a basis of future treatments aiming at the selective stabilization of tau-MT interactions.

scholarly journals Characterization of a New γTuRC Subunit with WD Repeats

2003 ◽  
Vol 14 (3) ◽  
pp. 1017-1026 ◽  
Author(s):  
Ruwanthi N. Gunawardane ◽  
Ona C. Martin ◽  
Yixian Zheng
Keyword(s):  
Microtubule Assembly ◽  
Guanine Nucleotide ◽  
Protein Subunits ◽  
Multiple Protein ◽  
Tubulin Binding ◽  
Ring Complex ◽  
Signature Sequences ◽  
Complete Set ◽  
Wd Repeats

The γ-tubulin ring complex (γTuRC), consisting of multiple protein subunits, can nucleate microtubule assembly. Although many subunits of the γTuRC have been identified, a complete set remains to be defined in any organism. In addition, how the subunits interact with each other to assemble into γTuRC remains largely unknown. Here, we report the characterization of a novel γTuRC subunit, Drosophila gamma ring protein with WD repeats (Dgp71WD). With the exception of γ-tubulin, Dgp71WD is the only γTuRC component identified to date that does not contain the grip motifs, which are signature sequences conserved in γTuRC components. By performing immunoprecipitations after pair-wise coexpression in Sf9 cells, we show that Dgp71WD directly interacts with the grip motif–containing γTuRC subunits, Dgrips84, 91, 128, and 163, suggesting that Dgp71WD may play a scaffolding role in γTuRC organization. We also show that Dgrips128 and 163, like Dgrips84 and 91, can interact directly with γ-tubulin. Coexpression of any of these grip motif–containing proteins with γ-tubulin promotes γ-tubulin binding to guanine nucleotide. In contrast, in the same assay Dgp71WD interacts with γ-tubulin but does not facilitate nucleotide binding.

Perinuclear localization of huntingtin as a consequence of its binding to microtubules through an interaction with β-tubulin: relevance to Huntington's disease

2002 ◽  
Vol 115 (5) ◽  
pp. 941-948 ◽  
Author(s):  
Guylaine Hoffner ◽  
Pascal Kahlem ◽  
Philippe Djian
Keyword(s):  
Electron Microscopy ◽  
Huntington's Disease ◽  
Huntington’S Disease ◽  
Binding Property ◽  
Perinuclear Region ◽  
Β Subunit ◽  
Tubulin Binding ◽  
Β Tubulin

Huntington's disease results from an expansion of a series of glutamine repeats in the protein huntingtin. We have discovered from immunopurification studies that huntingtin combines specifically with the β subunit of tubulin. This binding explains why huntingtin can be shown on assembled microtubules by electron microscopy. Immunostaining shows that most of the huntingtin in the cytoplasm is associated with microtubules. Huntingtin is particularly abundant in the perinuclear region, where it is also associated with microtubules and in the centrosomal region, where it co-localizes withγ-tubulin. In Huntington's disease, inclusions are often nuclear or perinuclear. Since the perinuclear concentration of huntingtin does not depend on the number of its glutamine repeats, we propose that inclusions are found in perinuclear and intranuclear locations because the β-tubulin binding property of huntingtin brings it to the perinuclear region, from which it readily gains access to the nucleus. The mutational glutamine expansion then promotes insolubility and results in an inclusion.

scholarly journals Mutations in the β-tubulin binding site for peloruside A confer resistance by targeting a cleft significant in side chain binding

Cell Cycle ◽  
2011 ◽  
Vol 10 (19) ◽  
pp. 3387-3396 ◽  
Author(s):  
Adrian Begaye ◽  
Shana Trostel ◽  
Zhiming Zhao ◽  
Richard E. Taylor ◽  
David C. Schriemer ◽  
...  
Keyword(s):  
Binding Site ◽  
Side Chain ◽  
Peloruside A ◽  
Tubulin Binding ◽  
Β Tubulin

scholarly journals CHK2–BRCA1 tumor-suppressor axis restrains oncogenic Aurora-A kinase to ensure proper mitotic microtubule assembly

2016 ◽  
Vol 113 (7) ◽  
pp. 1817-1822 ◽  
Author(s):  
Norman Ertych ◽  
Ailine Stolz ◽  
Oliver Valerius ◽  
Gerhard H. Braus ◽  
Holger Bastians
Keyword(s):  
Tumor Suppressor ◽  
Human Cancer ◽  
Microtubule Assembly ◽  
Cancer Type ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Mitotic Spindles ◽  
Human Cancer Cells ◽  
Chromosome Missegregation ◽  
Open Question

BRCA1 (breast cancer type 1 susceptibility protein) is a multifunctional tumor suppressor involved in DNA damage response, DNA repair, chromatin regulation, and mitotic chromosome segregation. Although the nuclear functions of BRCA1 have been investigated in detail, its role during mitosis is little understood. It is clear, however, that loss of BRCA1 in human cancer cells leads to chromosomal instability (CIN), which is defined as a perpetual gain or loss of whole chromosomes during mitosis. Moreover, our recent work has revealed that the mitotic function of BRCA1 depends on its phosphorylation by the tumor-suppressor kinase Chk2 (checkpoint kinase 2) and that this regulation is required to ensure normal microtubule plus end assembly rates within mitotic spindles. Intriguingly, loss of the positive regulation of BRCA1 leads to increased oncogenic Aurora-A activity, which acts as a mediator for abnormal mitotic microtubule assembly resulting in chromosome missegregation and CIN. However, how the CHK2–BRCA1 tumor suppressor axis restrains oncogenic Aurora-A during mitosis to ensure karyotype stability remained an open question. Here we uncover a dual molecular mechanism by which the CHK2–BRCA1 axis restrains oncogenic Aurora-A activity during mitosis and identify BRCA1 itself as a target for Aurora-A relevant for CIN. In fact, Chk2-mediated phosphorylation of BRCA1 is required to recruit the PP6C–SAPS3 phosphatase, which acts as a T-loop phosphatase inhibiting Aurora-A bound to BRCA1. Consequently, loss of CHK2 or PP6C-SAPS3 promotes Aurora-A activity associated with BRCA1 in mitosis. Aurora-A, in turn, then phosphorylates BRCA1 itself, thereby inhibiting the mitotic function of BRCA1 and promoting mitotic microtubule assembly, chromosome missegregation, and CIN.

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