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

Abstract

We present the first atomic model of a substrate-bound inner mitochondrial membrane AAA+ quality control protease, YME1. Our ~3.4 Å cryo-EM structure reveals how the ATPases form a closed spiral staircase encircling an unfolded substrate, directing it toward the flat, symmetric protease ring. Importantly, the structure reveals how 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 ATP hydrolysis cycle that results in step-wise substrate translocation. Furthermore, we identify a hinge-like linker that accommodates the large-scale nucleotide-driven motions of the ATPase spiral independently of the contiguous planar proteolytic base. These results define the first molecular mechanism for a mitochondrial inner membrane AAA+ protease and reveal a translocation mechanism likely conserved for other AAA+ ATPases.

Structures, Functions, and Dynamics of ESCRT-III/Vps4 Membrane Remodeling and Fission Complexes

“…This generalized model is supported by conservation of pore loop residues and other key structural elements (Monroe & Hill 2016, Sauer & Baker 2011) and by a series of recent structures of other AAA+ ATPases in complex with mixed polypeptide substrates (Deville et al 2017, Gates et al 2017, Puchades et al 2017, Ripstein et al 2017). These structures collectively represent a long-awaited breakthrough in our understanding of this large and important class of cellular machines (Harrison 2004), whose well-known representatives include essential activities like the 19S proteasome, NSF, and p97/Cdc48.…”

“…For example, the existing Vps4 structures do not obviously explain why ATP is hydrolyzed as subunits pass through the C/D transition even though the A–D subunits adopt similar conformations. A detailed mechanistic model for allosteric coupling was recently proposed on the basis of the structure of a nonhydrolyzing mutant of the AAA+ protease YME1 bound to ATP/ADP and a polypeptide substrate (Puchades et al 2017). This model is similar to that described above for Vps4 but differs in the proposed site of ATP hydrolysis (which is uncertain owing to use of an inactive enzyme for YME1 and a nonhydrolyzable nucleotide for Vps4).…”

Protein translocation by the SecA ATPase occurs by a power‐stroke mechanism

“…In this case, each of the six subunits has a loop analogous to the two-helix finger of SecA, which pushes the polypeptide chain through the central pore (Hinnerwisch et al, 2005;Martin et al, 2008;Han et al, 2017;Puchades et al, 2017;Ho et al, 2018). In this case, each of the six subunits has a loop analogous to the two-helix finger of SecA, which pushes the polypeptide chain through the central pore (Hinnerwisch et al, 2005;Martin et al, 2008;Han et al, 2017;Puchades et al, 2017;Ho et al, 2018).…”

“…Hexameric ATPases that move polypeptides, such as the 19S subunit of the proteasome, the Cdc48 ATPase, and the Clp proteins, may use a similar mechanism as SecA. In this case, each of the six subunits has a loop analogous to the two-helix finger of SecA, which pushes the polypeptide chain through the central pore (Hinnerwisch et al, 2005;Martin et al, 2008;Han et al, 2017;Puchades et al, 2017;Ho et al, 2018). Because it is difficult to separate the movements of the six loops during the ATP hydrolysis cycles, even with single-molecule experiments (Aubin-Tam et al, 2011;Sen et al, 2013;Olivares et al, 2014), monomeric SecA provides a unique, tractable model to determine the mechanism by which ATPases move polypeptides.…”

Coordinating Mitochondrial Biology Through the Stress-Responsive Regulation of Mitochondrial Proteases

“…These proteases all function through a similar biochemical mechanism that appears to be conserved from bacteria to mammals (Puchades et al, 2017; Sauer and Baker, 2011). Damaged and/or aberrantly-folded proteins are directed to the AAA+ domains of these proteases where they are unfolded through an ATP-dependent process (Puchades et al, 2017; Sauer and Baker, 2011). The unfolded proteins are then translocated to a protected proteolytic core for degradation.…”