%0 Journal Article %A Zev. A Ripstein %A Rui Huang %A Rafal Augustyniak %A Lewis E. Kay %A John L. Rubinstein %T Structure of a AAA+ unfoldase in the process of unfolding substrate %D 2017 %R 10.1101/105866 %J bioRxiv %P 105866 %X AAA+ unfoldases are thought to unfold substrate through the central pore of their hexameric structures, but how this process occurs is not known. VAT, the Thermoplasma acidophilum homologue of eukaryotic CDC48/p97, works in conjunction with the proteasome to degrade misfolded or damaged proteins. We show that in the presence of ATP, VAT with its regulatory N-terminal domains removed unfolds other VAT complexes as substrate. We captured images of this transient process by electron cryomicroscopy (cryo-EM) to reveal the structure of the substrate-bound intermediate. Substrate binding breaks the six-fold symmetry of the complex, allowing five of the six VAT subunits to constrict into a tight helix that grips an ~80 Å stretch of unfolded protein. The structure suggests a processive hand-over-hand unfolding mechanism, where each VAT subunit releases the substrate in turn before re-engaging further along the target protein, thereby unfolding it. %U https://www.biorxiv.org/content/biorxiv/early/2017/02/04/105866.full.pdf