We have established that Hsp104 employs distinct modes of intersubunit collaboration to resolve disordered aggregates versus amyloid. For disordered aggregates, Hsp104 subunits use probabilistic ATP hydrolysis similar to the mechanism defined for ClpX, a protein unfoldase (Martin et al., 2005
). However, unlike ClpX, Hsp104 withstands subunits that cannot bind substrate. ClpX hexamers are severely impaired by 2 subunits that cannot engage substrate (Martin et al., 2008
), whereas Hsp104 retains ~70% activity. This sensitivity might explain why ClpX is a poor protein disaggregase (Doyle et al., 2007a
The permissive nature of Hsp104 hexamers to subunits that cannot hydrolyze ATP or engage substrate enables a highly flexible disaggregase. Thus, 1 WT subunit per hexamer is sufficient to catalyze disaggregation (). Indeed, any opportunely positioned subunit within the hexamer that can hydrolyze ATP and engage the irregular and heterogeneous aggregated structure can promote disaggregation. Individual subunits do not have to co-ordinate ATPase or substrate-binding events with neighboring subunits or wait until all subunits are engaged, which may be sterically improbable. Thus, Hsp104 can resolve the unrelated proteins of the aggregated proteome after stress.
Mechanisms of intersubunit collaboration for Hsp104 and ClpB
Surprisingly, ClpB, the E. coli homolog of Hsp104, is tuned differently to Hsp104. Like Hsp104, ClpB exploits probabilistic substrate binding to dissolve disordered aggregates and tolerates subunits that cannot bind substrate (). This shared feature of ClpB and Hsp104 distinguishes them from the protein unfoldase, ClpX.
Unlike Hsp104, ClpB couples probabilistic substrate binding to highly co-operative ATP hydrolysis (). Unexpectedly, this operating mode enables ClpB to dissolve disordered aggregates more effectively than Hsp104. However, this enhancement comes at the expense of robust disaggregase activity able to accommodate ATPase-defective subunits. Unlike Hsp104, ClpB hexamers cannot tolerate a single ATPase-defective subunit. Our data also suggest that unlike Hsp104, ClpB has limited ability to couple cooperative ATPase activity to co-operative substrate handling, which is necessary to remodel amyloid.
The robustness and plasticity of Hsp104 hexamers is likely an adaptation that enables amyloid remodeling and empowers yeast to exploit prions for beneficial purposes. Indeed, ClpB and E. coli
cytosol were unable to remodel amyloid. Amyloid can accumulate in E. coli
upon protein overexpression (Wang et al., 2008
). Yet, ClpB’s limited amyloid-remodeling activity suggests that E. coli
compartmentalizes amyloid rather than disseminating it throughout the cytoplasm. Yeast also partition amyloid, but simultaneously disperse cytosolic prions for beneficial purposes. The profound selective advantages afforded by yeast prions are only made possible by Hsp104’s potent amyloid-remodeling activity (Alberti et al., 2009
; Halfmann et al., 2012
; Shorter and Lindquist, 2005
We suggest that Hsp104’s default intersubunit collaboration mechanism is probabilistic (). However, this default-operating mode can be rapidly retuned to a suitable subglobal or global co-operative mechanism upon sensing stable substrates. Thus, amyloid likely antagonizes unfolding and elicits a signal for Hsp104 subunits to work together to engage substrate, hydrolyze ATP and promote disaggregation (). For less chemically stable NM4 prions, a subglobal co-operative mechanism that is inactivated by 3 mutant subunits per hexamer is employed (). By contrast, NM25 prions, which are more stable and possess a longer cross-β core, are resolved by a global co-operative mechanism that is inactivated by 1 mutant subunit (). Ure2 prions and α-synA30P
amyloid are also resolved in this way (). Cryo-EM reconstructions indicate that Hsp104 might use a co-operative, sequential mechanism of substrate handling (Wendler et al., 2009
). However, we suggest that hexamer plasticity enables Hsp104 to adapt a variety of mechanochemical coupling mechanisms that are responsive to the specific physical demands of the aggregated substrate. Thus, Hsp104 is wired do the minimum work necessary to disaggregate any given substrate, i.e. if 2 subunits are sufficient to rapidly disaggregate a substrate, then only 2 will be used. Various multimeric, NTP-fueled ring-translocases with diverse substrate portfolios could use similar adaptable repertoires of intersubunit collaboration.
We establish that D704N or L462R mutations impair intersubunit communication, reduce plasticity and selectively ablate amyloid disaggregation. Indeed, D704 and L462 likely transmit or receive signals to recruit additional Hsp104 subunits during prion disaggregation (). Although further studies are needed to gain a structural understanding of how Hsp104 switches mechanism, our findings explain why Hsp104D704N
are functional in thermotolerance but defective in prion propagation (Kurahashi and Nakamura, 2007
Hsp104 might be designed to be more potent and selective against specific proteins, which could empower facile purification of irksome recombinant proteins for basic or therapeutic purposes. Hsp104 could also be developed to target select misfolded proteins in neurodegenerative disease (Vashist et al., 2010
). The intrinsic ability of Hsp104 to remodel diverse disease-associated amyloids as well as toxic oligomers suggests that this avenue warrants exploration. Here, it will be key to increase the specificity of the Hsp104 hexamer for a target polypeptide while simultaneously tuning plasticity such that toxic conformers are selectively eradicated. For example, hypomorphic scaffolds based on Hsp104D704N
could be useful in settings where amyloids are protective and disordered aggregates are toxic.