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

Abstract

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.

Defining the Molecular Basis of BubR1 Kinetochore Interactions and APC/C-CDC20 Inhibition

“…Such understanding requires detailed molecular information regarding BubR1 and the basis of its interactions both at the kinetochore and within the MCC. Thus far, this has not been possible as recombinant protein instability has hampered high resolution structural analysis of BubR1 or its paralogue Mad3 (35, 36). Here, we have overcome this difficulty and, through detailed mapping of the molecular architecture of the functionally significant N-terminal portion of human BubR1, have solved the crystal structure of its TPR domain at 1.8 Å resolution.…”

“…These links remain as conjecture as such mechanistic insight requires a detailed molecular understanding of BubR1. Molecular detail of BubR1 is not available as attempts to produce stable protein for high resolution structural studies have been hampered by BubR1 susceptibility to proteolysis, rendering such studies thus far unsuccessful (35, 36). …”

O-GlcNAc modulation at Akt1 Ser473 correlates with apoptosis of murine pancreatic β cells

“…Spots on silver-stained gels corresponding to Akt1 were excised, destained by reduction using 30 mM potassium ferricyanide/100 mM sodium thiosulfate, and washed with distilled water for MALDI-TOF/TOF analysis as described previously [19]. Briefly, gel pieces were incubated with 0.…”

Conservation of Helical Bundle Structure between the Exocyst Subunits

“…Limited proteolysis proved critical for most of the current exocyst structures; however, this strategy relies on availability of at least slightly soluble protein. An alternative approach is to computationally predict domain structures based on the similarity to proteins with known structural domains [20], [22]–[24]. However, this approach is challenging if the protein has little or no similarity with proteins of known structures.…”