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1.
J Biol Chem ; 295(6): 1517-1538, 2020 02 07.
Article in English | MEDLINE | ID: mdl-31882541

ABSTRACT

Hsp104 is a hexameric AAA+ ring translocase, which drives protein disaggregation in nonmetazoan eukaryotes. Cryo-EM structures of Hsp104 have suggested potential mechanisms of substrate translocation, but precisely how Hsp104 hexamers disaggregate proteins remains incompletely understood. Here, we employed synchrotron X-ray footprinting to probe the solution-state structures of Hsp104 monomers in the absence of nucleotide and Hsp104 hexamers in the presence of ADP or ATPγS (adenosine 5'-O-(thiotriphosphate)). Comparing side-chain solvent accessibilities between these three states illuminated aspects of Hsp104 structure and guided design of Hsp104 variants to probe the disaggregase mechanism in vitro and in vivo We established that Hsp104 hexamers switch from a more-solvated state in ADP to a less-solvated state in ATPγS, consistent with switching from an open spiral to a closed ring visualized by cryo-EM. We pinpointed critical N-terminal domain (NTD), NTD-nucleotide-binding domain 1 (NBD1) linker, NBD1, and middle domain (MD) residues that enable intrinsic disaggregase activity and Hsp70 collaboration. We uncovered NTD residues in the loop between helices A1 and A2 that can be substituted to enhance disaggregase activity. We elucidated a novel potentiated Hsp104 MD variant, Hsp104-RYD, which suppresses α-synuclein, fused in sarcoma (FUS), and TDP-43 toxicity. We disambiguated a secondary pore-loop in NBD1, which collaborates with the NTD and NBD1 tyrosine-bearing pore-loop to drive protein disaggregation. Finally, we defined Leu-601 in NBD2 as crucial for Hsp104 hexamerization. Collectively, our findings unveil new facets of Hsp104 structure and mechanism. They also connect regions undergoing large changes in solvation to functionality, which could have profound implications for protein engineering.


Subject(s)
Heat-Shock Proteins/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/chemistry , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/metabolism , Heat-Shock Proteins/metabolism , Models, Molecular , Protein Aggregates , Protein Conformation , Protein Multimerization , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Synchrotrons , X-Rays
2.
Cell Rep ; 28(8): 2080-2095.e6, 2019 08 20.
Article in English | MEDLINE | ID: mdl-31433984

ABSTRACT

Hsp104 is an AAA+ protein disaggregase, which can be potentiated via diverse mutations in its autoregulatory middle domain (MD) to mitigate toxic misfolding of TDP-43, FUS, and α-synuclein implicated in fatal neurodegenerative disorders. Problematically, potentiated MD variants can exhibit off-target toxicity. Here, we mine disaggregase sequence space to safely enhance Hsp104 activity via single mutations in nucleotide-binding domain 1 (NBD1) or NBD2. Like MD variants, NBD variants counter TDP-43, FUS, and α-synuclein toxicity and exhibit elevated ATPase and disaggregase activity. Unlike MD variants, non-toxic NBD1 and NBD2 variants emerge that rescue TDP-43, FUS, and α-synuclein toxicity. Potentiating substitutions alter NBD1 residues that contact ATP, ATP-binding residues, or the MD. Mutating the NBD2 protomer interface can also safely ameliorate Hsp104. Thus, we disambiguate allosteric regulation of Hsp104 by several tunable structural contacts, which can be engineered to spawn enhanced therapeutic disaggregases with minimal off-target toxicity.


Subject(s)
DNA-Binding Proteins/toxicity , Heat-Shock Proteins/metabolism , RNA-Binding Protein FUS/toxicity , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , alpha-Synuclein/toxicity , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Azetidinecarboxylic Acid/pharmacology , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/genetics , Mutant Proteins/metabolism , Mutation, Missense/genetics , Protein Aggregates , Protein Domains , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Temperature
3.
Nature ; 532(7600): 527-30, 2016 Apr 28.
Article in English | MEDLINE | ID: mdl-27042935

ABSTRACT

The human σ1 receptor is an enigmatic endoplasmic-reticulum-resident transmembrane protein implicated in a variety of disorders including depression, drug addiction, and neuropathic pain. Recently, an additional connection to amyotrophic lateral sclerosis has emerged from studies of human genetics and mouse models. Unlike many transmembrane receptors that belong to large, extensively studied families such as G-protein-coupled receptors or ligand-gated ion channels, the σ1 receptor is an evolutionary isolate with no discernible similarity to any other human protein. Despite its increasingly clear importance in human physiology and disease, the molecular architecture of the σ1 receptor and its regulation by drug-like compounds remain poorly defined. Here we report crystal structures of the human σ1 receptor in complex with two chemically divergent ligands, PD144418 and 4-IBP. The structures reveal a trimeric architecture with a single transmembrane domain in each protomer. The carboxy-terminal domain of the receptor shows an extensive flat, hydrophobic membrane-proximal surface, suggesting an intimate association with the cytosolic surface of the endoplasmic reticulum membrane in cells. This domain includes a cupin-like ß-barrel with the ligand-binding site buried at its centre. This large, hydrophobic ligand-binding cavity shows remarkable plasticity in ligand recognition, binding the two ligands in similar positions despite dissimilar chemical structures. Taken together, these results reveal the overall architecture, oligomerization state, and molecular basis for ligand recognition by this important but poorly understood protein.


Subject(s)
Receptors, sigma/chemistry , Benzamides/chemistry , Benzamides/metabolism , Binding Sites , Crystallography, X-Ray , Endoplasmic Reticulum/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Intracellular Membranes/metabolism , Isoxazoles/chemistry , Isoxazoles/metabolism , Ligands , Models, Molecular , Piperidines/chemistry , Piperidines/metabolism , Protein Structure, Tertiary , Pyridines/chemistry , Pyridines/metabolism , Receptors, sigma/metabolism , Substrate Specificity , Sigma-1 Receptor
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