Structural analysis of Bub3 interactions in the mitotic spindle checkpoint.

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• The transition from metaphase to anaphase in cell division is regulated by key proteins, including Mad3/BubR1, Mad2, Bub1, and Bub3.
• Mad3 from Saccharomyces cerevisiae shares homology with Bub1 but lacks a kinase domain, forming a stable heterodimer with Bub3.

Purpose of the Study:

• To investigate the structural basis of the interaction between Mad3 and Bub3.
• To elucidate the role of Gle2-binding-sequence (GLEBS) motifs in Mad3-Bub3 complex formation and function.
• To understand how this complex is recruited to kinetochores for cell cycle regulation.

Main Methods:

• Negative-stain electron microscopy to determine the molecular structure of Mad3 and Bub3.
• Calorimetry to quantify the binding affinity between GLEBS-motif peptides and Bub3.
• X-ray crystallography to obtain high-resolution structures of GLEBS peptides bound to Bub3.

Main Results:

• Mad3 is an extended molecule, while Bub3 is globular; they form a stable heterodimer.
• GLEBS motifs in Mad3 and Bub1 are necessary and sufficient for Bub3 interaction, with dissociation constants around 5 microM.
• Crystal structures reveal similar, mutually exclusive binding of Mad3 and Bub1 GLEBS peptides to Bub3's beta-propeller surface.
• Mutations disrupting the interface lead to checkpoint deficiency and chromosome instability.

Conclusions:

• The structural interaction between GLEBS motifs and Bub3 is critical for Mad3/Bub1 function in the spindle assembly checkpoint.
• The specific structure of the GLEBS segment, stabilized by Bub3, facilitates recruitment to unattached kinetochores.
• This mechanism ensures proper chromosome segregation and genomic stability during cell division.