Structure of the mitochondrial inner membrane AAA+ protease YME1 gives insight into substrate processing

INTRODUCTION Protein quality control is essential for mitochondrial function, and imbalances in this regulation are associated with numerous human diseases. YME1L is a hexameric AAA+ protease in the inner membrane (IM) that controls maintenance of the electron transport chain, protein import, lipid synthesis, and mitochondrial morphology. Every YME1L subunit contains an adenosine triphosphatase (ATPase) and a peptidase domain, which reside in the intermembrane space, tethered to the IM by a membrane helix. Protein substrates undergo adenosine triphosphate (ATP)–driven translocation through a central pore into a proteolytic chamber by a mechanism that is likely to be conserved in other AAA+ proteases. RATIONALE A compelling question is how YME1L couples ATP hydrolysis to processive substrate translocation. We sought to understand this mechanism by determining a near–atomic resolution cryo–electron microscopy (cryo-EM) structure of a solubilized form of the yeast homolog, YME1. By precisely visualizing how the nucleotide state of YME1 subunits allosterically controls their interaction with a translocating protein substrate, we can understand how cycles of nucleotide hydrolysis can drive stepwise translocation of protein substrates for degradation. RESULTS Our ~3.4 A resolution structure shows that YME1 assembles into two stacked rings, with an asymmetric spiral staircase of ATPase domains atop a planar protease ring. Tyrosine residues in two conserved central ATPase pore loops grasp an unfolded 10–amino acid peptide and direct it toward a negatively charged proteolytic chamber. The four central subunits in the staircase bind ATP and intercalate their pore loop tyrosines with the substrate backbone in a configuration compatible with sequence-independent translocation. The lowest “posthydrolysis” subunit contains an adenosine diphosphate (ADP)–like EM density and only interacts modestly with the substrate, whereas the top apo-like step subunit does not contain well-resolved nucleotide density and is disengaged from both the substrate and the ATPase ring. Bound ATP is sensed by the adjacent protomer via two arginine fingers and an intersubunit signaling (ISS) motif that bridges the subunits across the nucleotide-binding pocket. Attachment of the ISS positions the pore loop tyrosines of the ATP-bound subunit to tightly grasp the substrate. Loss of the γ-phosphate releases the arginine fingers, retracting the ISS and repositioning the pore loops away from the substrate. The absence of nucleotide in the step subunit breaks coordination on both sides and sequesters the pore loops into helices away from the substrate. A glycine residue in the interdomain linker is required to accommodate large movements of the ATPase domains within the spiral staircase. CONCLUSION This structure of a substrate-bound single-polypeptide AAA+ protease allows us to define a tightly coordinated sequential ATP hydrolysis cycle. Hydrolysis in the lowest ATP-bound subunit abolishes coordination by the adjacent arginine fingers and ISS, repositioning the now posthydrolysis subunit to the lowest position of the staircase, which, in turn, triggers hydrolysis in the next-lowest ATP-bound subunit. Loss of coordination on both sides of the ADP-bound subunit breaks substrate interaction and displaces the subunit from the hexamer, where it can release ADP and rebind ATP at the top of the staircase. Iteration of this cycle drives stepwise translocation of the substrate into the proteolytic chamber. The high degree of structural conservation between YME1 and the 26 S proteasome suggests that this mechanism may be conserved across ATP-driven proteases.