Structural and mechanistic insights into Hsp104 function revealed by synchrotron X-ray footprinting. J Biol Chem 2020 Feb 07;295(6):1517-1538
Date
12/29/2019Pubmed ID
31882541Pubmed Central ID
PMC7008382DOI
10.1074/jbc.RA119.011577Scopus ID
2-s2.0-85079204211 (requires institutional sign-in at Scopus site) 11 CitationsAbstract
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.
Author List
Sweeny EA, Tariq A, Gurpinar E, Go MS, Sochor MA, Kan ZY, Mayne L, Englander SW, Shorter JAuthor
Elizabeth Sweeny PhD Assistant Professor in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Adenosine TriphosphateHeat-Shock Proteins
Models, Molecular
Protein Aggregates
Protein Conformation
Protein Multimerization
Saccharomyces cerevisiae
Saccharomyces cerevisiae Proteins
Synchrotrons
X-Rays
