Mechanism for remodelling of the cell cycle checkpoint protein MAD2 by the ATPase TRIP13

The spindle assembly checkpoint is a safeguard mechanism that coordinates cell-cycle progression during mitosis with the state of chromosome attachment to the mitotic spindle. The checkpoint prevents mitotic cells from exiting mitosis in the presence of unattached or improperly attached chromosomes, thus avoiding whole-chromosome gains or losses and their detrimental effects on cell physiology. Here, I review a considerable body of recent progress in the elucidation of the molecular mechanisms underlying checkpoint signaling, and identify a number of unresolved questions.

Hsp100 polypeptide translocases are conserved members of the AAA+ family (adenosine triphosphatases associated with diverse cellular activities) that maintain proteostasis by unfolding aberrant and toxic proteins for refolding or proteolytic degradation. The Hsp104 disaggregase from Saccharomyces cerevisiae solubilizes stress-induced amorphous aggregates and amyloids. The structural basis for substrate recognition and translocation is unknown. Using a model substrate (casein), we report cryo-electron microscopy structures at near-atomic resolution of Hsp104 in different translocation states. Substrate interactions are mediated by conserved, pore-loop tyrosines that contact an 80-angstrom-long unfolded polypeptide along the axial channel. Two protomers undergo a ratchet-like conformational change that advances pore loop-substrate interactions by two amino acids. These changes are coupled to activation of specific nucleotide hydrolysis sites and, when transmitted around the hexamer, reveal a processive rotary translocation mechanism and substrate-responsive flexibility during Hsp104-catalyzed disaggregation.

The formal description of a workflow to cryo-EM structure determination in the RELION program allows standardization of procedures and on-the-fly image processing during data acquisition.