Unfolding the mechanism of the AAA+ unfoldase VAT by a combined cryo-EM, solution NMR study

Abstract

The AAA+ (ATPases associated with a variety of cellular activities) enzymes play critical roles in a variety of homeostatic processes in all kingdoms of life. Valosin-containing protein-like ATPase of Thermoplasma acidophilum (VAT), the archaeal homolog of the ubiquitous AAA+ protein Cdc48/p97, functions in concert with the 20S proteasome by unfolding substrates and passing them on for degradation. Here, we present electron cryomicroscopy (cryo-EM) maps showing that VAT undergoes large conformational rearrangements during its ATP hydrolysis cycle that differ dramatically from the conformational states observed for Cdc48/p97. We validate key features of the model with biochemical and solution methyl-transverse relaxation optimized spectroscopY (TROSY) NMR experiments and suggest a mechanism for coupling the energy of nucleotide hydrolysis to substrate unfolding. These findings illustrate the unique complementarity between cryo-EM and solution NMR for studies of molecular machines, showing that the structural properties of VAT, as well as the population distributions of conformers, are similar in the frozen specimens used for cryo-EM and in the solution phase where NMR spectra are recorded.T he AAA+ enzymes (ATPases associated with a variety of cellular activities) use the energy of ATP hydrolysis to carry out a myriad of different biological functions that are critical to cellular homeostasis. These molecular machines are typically barrel-shaped, containing a narrow channel that serves to traffic substrates through the enzyme (1-4). In the case of the heat shock protein 104 (Hsp104) AAA+ class of chaperone, for example, substrates are dissociated from aggregates via a pulling motion that forces individual polypeptide chains through the central pore of the enzyme (1, 5). The unfolded proteins that emerge subsequently refold spontaneously or are acted on by additional chaperones that aid in their refolding (6). Other AAA+ enzymes act synergistically with proteases, unfolding targeted substrates and passing them directly to the proteases, where they are subsequently degraded (7). In this way, proteins that have become damaged or that are no longer required can be removed before their accumulation disrupts cellular function (8). A variety of different AAA+ unfoldases have been characterized, including Rpt1-6 in the 19S proteasome regulatory particle in eukaryotes (9-11), PAN in archaea (12-14), Mpa/ARC in Actinobacteria (15), ClpA/X (2) and HslU (3) in bacteria, and VAT in the Thermoplasma acidophilum archaebacterium (16)(17)(18)(19)(20).VAT is an ∼500-kDa homohexamer, with each monomer containing two tandem AAA+ domains, referred to as D1 and D2, and an N-terminal domain (NTD) distinct from the NTD of other AAA+ enzymes (16) (Fig. 1A). Both D1 and D2 are homologous to the single AAA+ modules of PAN and Rpt1-6 (1, 16). Biochemical studies have shown that VAT unfolds globular proteins and interacts directly with the 20S core particle (CP) of the proteasome, leading to enhanced proteolytic activity for both folded and ...

Structure of a AAA+ unfoldase in the process of unfolding substrate

“…The maps are similar up to 8 Å resolution, indicating that at least to this resolution the conformations of ΔN-VAT and full-length VAT are the same. ( f ) Local resolution estimation with ResMap (Kucukelbir et al, 2014) for the stacked-ring (left) and substrate-bound conformations (right). DOI: http://dx.doi.org/10.7554/eLife.25754.006

10.7554/eLife.25754.007Figure 2—figure supplement 2.Pore loops of VAT.Compared to the stacked-ring conformation (left) and the split-ring conformation determined previously (Huang et al, 2016) (right), the channel through the lumen of the substrate-engaged state of VAT (middle) formed by pore loop residues in both NBD1 ( a ) and NBD2 ( b ) is much more constrained. The pore loops of the seam protomer do not participate in forming the channel (gold).

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Molecular Mechanism of Substrate Processing by the Cdc48 ATPase Complex

“…Archaeal Cdc48 and some mammalian p97 mutants can also translocate polypeptides into associated 20S proteasomes under certain conditions (Barthelme and Sauer, 2012; 2013), implying movement through the central pore. However, archaeal Cdc48 undergoes conformational changes that have never been observed with eukaryotic homologs (Huang et al, 2016), and wild type yeast Cdc48 and mammalian p97 are thought not to use a translocation process (Banerjee et al, 2016; DeLaBarre and Brunger, 2005). A mechanism that does not involve translocation is also employed by Cdc48’s closest relative, the NEM-sensitive fusion protein (NSF) (Zhao et al, 2015).…”

Structures of biomolecular complexes by combination of NMR and cryoEM methods

“…As these polypeptides are often part of large biological complexes, NMR is instrumental at uncovering conformation changes or even folding events associated with assembly of the large complexes whose atomic structures were determined by cryoEM. NMR is also a unique approach to identify a distribution of conformers within a large molecular machine in solution [52]. By correlating the local structural heterogeneity observed by NMR and the different large scale rearrangements identified by cryoEM, it is possible to propose detailed pseudo-atomic models for these molecular machines in different conformational states.…”