Research ArticleStructural Biology

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

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Science  03 Nov 2017:
Vol. 358, Issue 6363, eaao0464
DOI: 10.1126/science.aao0464

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Feeding a protease step by step

Proteins that degrade damaged or misfolded mitochondrial proteins are essential for mitochondrial function. A key player is the hexameric protease YME1, in which each subunit is anchored in the inner mitochondrial membrane by a helix and has an adenosine triphosphatase (ATPase) domain and a protease domain in the intermembrane space. Puchades et al. report a high-resolution structure that shows that the ATPase domains form an asymmetric spiral staircase that stacks above a planar protease ring. Conserved tyrosine residues in the central pore of the spiral staircase interact with a substrate peptide. The ATP hydrolysis cycle is sequential and coordinated with changes in the position of the tyrosine residues that result in stepwise translocation of the substrate into the protease chamber.

Science, this issue p. eaao0464

Structured Abstract

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 Å 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 26S proteasome suggests that this mechanism may be conserved across ATP-driven proteases.

Cryo-EM structure of AAA+ protease YME1 sheds light on the mechanism of substrate translocation.

(1) A semitransparent surface representation shows how the asymmetric ATPase staircase is positioned above a planar C6-symmetric protease ring. Substrate (orange) surrounded by pore loop 1 tyrosines is shown in blue. Nucleotides are shown as gray densities. (2) A close-up view of the cryo-EM density reveals a spiral staircase organization with tyrosines intercalating into the substrate. (3) The nucleotide state could be identified for each subunit. (4) A cartoon representation of the YME1 ATPase hexamer depicts the asymmetric organization of the subunits surrounding the substrate. The ISS motif (represented as a Phe residue) protrudes into the nucleotide-binding pocket of the neighboring subunit only in the presence of ATP.

Abstract

We present an atomic model of a substrate-bound inner mitochondrial membrane AAA+ quality control protease in yeast, YME1. Our ~3.4-angstrom cryo–electron microscopy structure reveals how the adenosine triphosphatases (ATPases) form a closed spiral staircase encircling an unfolded substrate, directing it toward the flat, symmetric protease ring. Three coexisting nucleotide states allosterically induce distinct positioning of tyrosines in the central channel, resulting in substrate engagement and translocation to the negatively charged proteolytic chamber. This tight coordination by a network of conserved residues defines a sequential, around-the-ring adenosine triphosphate hydrolysis cycle that results in stepwise substrate translocation. A hingelike linker accommodates the large-scale nucleotide-driven motions of the ATPase spiral relative to the planar proteolytic base. The translocation mechanism is likely conserved for other AAA+ ATPases.

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