Crystal Structure of the Spindle Assembly Checkpoint Protein Bub3

Larsen, Nicholas

doi:10.1016/j.jmb.2004.09.094

Cited by 36 publications

(34 citation statements)

References 50 publications

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“…We have shown previously that apo-Bub3 is a canonical seven-bladed ␤-propeller (8). Each blade is a four-stranded ␤-sheet arranged in pseudo7-fold rotational symmetry about a central axis ( Fig.…”

Section: Mad3 Is An Extended Molecule With Predicted Tetratricopeptidementioning

confidence: 99%

“…Bub3 and Rae1 have 34% sequence identity and 52% similarity in humans (8). Although Bub3 appears to be specific for the GLEBS sequences of Bub1 and Mad3, murine Rae1 (mRae1) has been reported to bind the GLEBS motifs of mNup98 as well as mBub1 and mBubR1 (13).…”

Section: Mad3 Is An Extended Molecule With Predicted Tetratricopeptidementioning

confidence: 99%

“…This Gle2-Nup98 complex specifically inhibits APC Cdh1 -mediated ubiquitination of securin (15). Gle2/Rae1 and Bub3 are both WD40 proteins with significant homology to each other, and we therefore anticipate that the interactions between Nup98-Gle2 will resemble those between Mad3-Bub3 (8).…”

mentioning

confidence: 98%

“…Mad3 presumably originated from a duplicated copy of Bub1 and subsequently evolved a new or related function that did not require kinase activity. Mad3 and Bub1 both form constitutive complexes with Bub3 throughout the cell cycle, suggesting that this interaction is important for the function of both proteins (7,8). Primitive fungi that did not undergo whole-genome duplication have a Bub1 gene but lack a corresponding Mad3 gene.…”

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confidence: 99%

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Structural analysis of Bub3 interactions in the mitotic spindle checkpoint

Larsen

Al-Bassam

Wei

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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130

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The Mad3/BubR1, Mad2, Bub1, and Bub3 proteins are gatekeepers for the transition from metaphase to anaphase. Mad3 from Saccharomyces cerevisiae has homology to Bub1 but lacks a corresponding C-terminal kinase domain. Mad3 forms a stable heterodimer with Bub3. Negative-stain electron microscopy shows that Mad3 is an extended molecule (Ϸ200 Å long), whereas Bub3 is globular. The Gle2-binding-sequence (GLEBS) motifs found in Mad3 and Bub1 are necessary and sufficient for interaction with Bub3. The calorimetrically determined dissociation constants for GLEBS-motif peptides and Bub3 are Ϸ5 M. Crystal structures of these peptides with Bub3 show that the interactions for Mad3 and Bub1 are similar and mutually exclusive. In both structures, the GLEBS peptide snakes along the top surface of the ␤-propeller, forming an extensive interface. Mutations in either protein that disrupt the interface cause checkpoint deficiency and chromosome instability. We propose that the structure imposed on the GLEBS segment by its association with Bub3 enables recruitment to unattached kinetochores.␤-propeller ͉ crystal structures ͉ Rae1/Nup98 ͉ GLEBS motif

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Section: Mad3 Is An Extended Molecule With Predicted Tetratricopeptidementioning

confidence: 99%

Section: Mad3 Is An Extended Molecule With Predicted Tetratricopeptidementioning

confidence: 99%

mentioning

confidence: 98%

mentioning

confidence: 99%

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Structural analysis of Bub3 interactions in the mitotic spindle checkpoint

Larsen

Al-Bassam

Wei

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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130

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“…The three-dimensional structures of the checkpoint proteins, Mad1, Mad2 [22,23], and Saccharomyces cerevisiae Bub3p [24], have been reported in previous works. The structures of BubR1 and its homolog-proteins, however, have not yet been reported.…”

mentioning

confidence: 77%

Identification and purification of a soluble region of BubR1: A critical component of the mitotic checkpoint complex

Yoon

Kang

Kim

et al. 2005

Protein Expression and Purification

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The mitotic checkpoint complex (MCC) ensures the Wdelity of chromosomal segregation, by delaying the onset of anaphase until all sister chromatids have been properly attached to the mitotic spindle. In essence, this MCC-induced delay is achieved via the inhibition of the anaphase-promoting complex (APC). Among the components of the MCC, BubR1 plays two major roles in the functions of the mitotic checkpoint. First, BubR1 is able to inhibit APC activity, either by itself or as a component of the MCC, by sequestering a APC coactivator, known as Cdc20. Second, BubR1 activates mitotic checkpoint signaling cascades by binding to the centromere-associated protein E, a microtubule motor protein. Obtaining highly soluble BubR1 is a prerequisite for the study of its structure. BubR1 is a multi-domain protein, which includes a KEN box motif, a mad3-like region, a Bub3 binding domain, and a kinase domain. We obtained a soluble BubR1 construct using a three-step expression strategy. First, we obtained two constructs from BLAST sequence homology searches, both of which were expressed abundantly in the inclusion bodies. We then adjusted the lengths of the two constructs by secondary structure prediction, thereby generating partially soluble constructs. Third, we optimized the solubility of the two constructs by either chopping or adding a few residues at the C-terminus. Finally, we obtained a highly soluble BubR1 construct via the Escherichia coli expression system, which allowed for a yield of 10.8 mg/L culture. This report may provide insight into the design of highly soluble constructs of insoluble multi-domain proteins.  2005 Elsevier Inc. All rights reserved.Keywords: BubR1; Structure; Anaphase-promoting complex In the eukaryotic cell cycle, mitosis consists of six sequential phases: prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis. During metaphase, two sister chromatids align in the equatorial plane of the dividing cell, while mitotic spindles attach themselves to the kinetochores in the centromeres of the sister chromatids. The mitotic spindle-attached sister chromatids are then separated, and pulled into each spindle pole, thereby completing chromosomal duplication. The mitotic checkpoint (also called the spindle assembly checkpoint) ensures the Wdelity of this chromosomal segregation, by delaying the onset of anaphase until all sister chromatids have been properly attached to the mitotic spindle [1]. Bub3 [5], and BubR1 [6,7] are some characteristic mitotic checkpoint proteins. These proteins associate with the unattached kinetochores and are involved in the delay in the metaphase/ anaphase transition [4,5,[7][8][9]. In essence, this delay occurs due to inhibition of the anaphase-promoting * Corresponding author. Fax: +82 2 741 7947.

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Kinetochore‐catalyzed MCC formation: A structural perspective

Fischer

2022

IUBMB Life

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The spindle assembly checkpoint (SAC) is a cellular surveillance mechanism that functions to ensure accurate chromosome segregation during mitosis. Macromolecular complexes known as kinetochores, act as the interface of sister chromatid attachment to spindle microtubules. In response to unattached kinetochores, the SAC activates its effector, the mitotic checkpoint complex (MCC), which delays mitotic exit until all sister chromatid pairs have achieved successful attachment to the bipolar mitotic spindle. Formation of the MCC (composed of Mad2, BubR1, Bub3 and Cdc20) is regulated by an Mps1 kinase‐dependent phosphorylation signaling cascade which assembles and repositions components of the MCC onto a catalytic scaffold. This scaffold functions to catalyze the conversion of the HORMA‐domain protein Mad2 from an “inactive” open‐state (O‐Mad2) into an “active” closed‐Mad2 (C‐Mad2), and simultaneous Cdc20 binding. Here, our current understanding of the molecular mechanisms underlying the kinetic barrier to C‐Mad2:Cdc20 formation will be reviewed. Recent progress in elucidating the precise molecular choreography orchestrated by the catalytic scaffold to rapidly assemble the MCC will be examined, and unresolved questions will be highlighted. Ultimately, understanding how the SAC rapidly activates the checkpoint not only provides insights into how cells maintain genomic integrity during mitosis, but also provides a paradigm for how cells can utilize molecular switches, including other HORMA domain‐containing proteins, to make rapid changes to a cell's physiological state.

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Crystal Structure of the Spindle Assembly Checkpoint Protein Bub3

Cited by 36 publications

References 50 publications

Structural analysis of Bub3 interactions in the mitotic spindle checkpoint

Structural analysis of Bub3 interactions in the mitotic spindle checkpoint

Identification and purification of a soluble region of BubR1: A critical component of the mitotic checkpoint complex

Kinetochore‐catalyzed MCC formation: A structural perspective

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