Hsp100 polypeptide translocases are conserved AAA+ machines that maintain proteostasis by unfolding aberrant and toxic proteins for refolding or proteolytic degradation. The Hsp104 disaggregase from S. cerevisiae solubilizes stress-induced amorphous aggregates and amyloid. The structural basis for substrate recognition and translocation is unknown. Using a model substrate (casein), we report cryo-EM structures at near-atomic resolution of Hsp104 in different translocation states. Substrate interactions are mediated by conserved, pore-loop tyrosines that contact an 80 Å-long unfolded polypeptide along the axial channel. Two protomers undergo a ratchet-like conformational change that advances pore-loop-substrate interactions by two-amino acids. These changes are coupled to activation of specific ATPase sites and, when transmitted around the hexamer, reveal a processive rotary translocation mechanism and a remarkable structural plasticity of Hsp104-catalyzed disaggregation.

ClpB and Hsp104 are conserved AAA+ protein disaggregases that promote survival during cellular stress. Hsp104 acts on amyloids, supporting prion propagation in yeast, and can solubilize toxic oligomers connected to neurodegenerative diseases. A definitive structural mechanism, however, has remained elusive. We have determined the cryo-EM structure of Hsp104 in the ATP state, revealing a near-helical hexamer architecture that coordinates the mechanical power of the twelve AAA+ domains for disaggregation. An unprecedented heteromeric AAA+ interaction defines an asymmetric seam in an apparent catalytic arrangement that aligns the domains in a two-turn spiral. N-terminal domains interact to form a broad channel entrance for substrate engagement and Hsp70 interaction. Middle-domain helices bridge adjacent protomers across the nucleotide pocket, explaining roles in hydrolysis and disaggregation. Remarkably, substrate-binding pore loops line the channel in a continuous spiral that appears optimized for substrate transfer across the AAA+ domains, establishing a directional path for polypeptide translocation.

Highlights d Mining Hsp104 sequence space to safely enhance disaggregase activity d Non-toxic NBD1 and NBD2 variants counter TDP-43, FUS, and a-synuclein toxicity d Mutating NBD1 residues that engage ATP or ATP-binding residues potentiates activity d Mutating the NBD2 protomer interface can safely ameliorate Hsp104 activity

Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution.

Hsp104 is a hexameric AAA + ATPase and protein disaggregase found in yeast, which can be potentiated via mutations in its middle domain (MD) to counter toxic phase separation by TDP-43, FUS and α-synuclein connected to devastating neurodegenerative disorders. Subtle missense mutations in the Hsp104 MD can enhance activity, indicating that post-translational modification of specific MD residues might also potentiate Hsp104. Indeed, several serine and threonine residues throughout Hsp104 can be phosphorylated in vivo. Here, we introduce phosphomimetic aspartate or glutamate residues at these positions and assess Hsp104 activity. Remarkably, phosphomimetic T499D/E and S535D/E mutations in the MD enable Hsp104 to counter TDP-43, FUS and α-synuclein aggregation and toxicity in yeast, whereas T499A/V/I and S535A do not. Moreover, Hsp104T499E and Hsp104S535E exhibit enhanced ATPase activity and Hsp70-independent disaggregase activity in vitro. We suggest that phosphorylation of T499 or S535 may elicit enhanced Hsp104 disaggregase activity in a reversible and regulated manner.

Cells have evolved a sophisticated proteostasis network to ensure that proteins acquire and retain their native structure and function. Critical components of this network include molecular chaperones and protein disaggregases, which function to prevent and reverse deleterious protein misfolding. Nevertheless, proteostasis networks have limits, which when exceeded can have fatal consequences as in various neurodegenerative disorders, including Parkinson's disease and amyotrophic lateral sclerosis. A promising strategy is to engineer proteostasis networks to counter challenges presented by specific diseases or specific proteins. Here, we review efforts to enhance the activity of individual molecular chaperones or protein disaggregases via engineering and directed evolution. Remarkably, enhanced global activity or altered substrate specificity of various molecular chaperones, including GroEL, Hsp70, ClpX, and Spy, can be achieved by minor changes in primary sequence and often a single missense mutation. Likewise, small changes in the primary sequence of Hsp104 yield potentiated protein disaggregases that reverse the aggregation and buffer toxicity of various neurodegenerative disease proteins, including α-synuclein, TDP-43, and FUS. Collectively, these advances have revealed key mechanistic and functional insights into chaperone and disaggregase biology. They also suggest that enhanced chaperones and disaggregases could have important applications in treating human disease as well as in the purification of valuable proteins in the pharmaceutical sector.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease marked by progressive loss of muscle function. It is the most common adult‐onset form of motor neuron disease, affecting about 16 000 people in the United States alone. The average survival is about 3 years. Only two interventional drugs, the antiglutamatergic small‐molecule riluzole and the more recent antioxidant edaravone, have been approved for the treatment of ALS to date. Therapeutic strategies under investigation in clinical trials cover a range of different modalities and targets, and more than 70 different drugs have been tested in the clinic to date. Here, we summarize and classify interventional therapeutic strategies based on their molecular targets and phenotypic effects. We also discuss possible reasons for the failure of clinical trials in ALS and highlight emerging preclinical strategies that could provide a breakthrough in the battle against this relentless disease.

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