Overlapping and Specific Functions of the Hsp104 N Domain Define Its Role in Protein Disaggregation

Abstract

Hsp104 is a ring-forming protein disaggregase that rescues stress-damaged proteins from an aggregated state. To facilitate protein disaggregation, Hsp104 cooperates with Hsp70 and Hsp40 chaperones (Hsp70/40) to form a bi-chaperone system. How Hsp104 recognizes its substrates, particularly the importance of the N domain, remains poorly understood and multiple, seemingly conflicting mechanisms have been proposed. Although the N domain is dispensable for protein disaggregation, it is sensitive to point mutations that abolish the function of the bacterial Hsp104 homolog in vitro, and is essential for curing yeast prions by Hsp104 overexpression in vivo. Here, we present the crystal structure of an N-terminal fragment of Saccharomyces cerevisiae Hsp104 with the N domain of one molecule bound to the C-terminal helix of the neighboring D1 domain. Consistent with mimicking substrate interaction, mutating the putative substrate-binding site in a constitutively active Hsp104 variant impairs the recovery of functional protein from aggregates. We find that the observed substrate-binding defect can be rescued by Hsp70/40 chaperones, providing a molecular explanation as to why the N domain is dispensable for protein disaggregation when Hsp70/40 is present, yet essential for the dissolution of Hsp104-specific substrates, such as yeast prions, which likely depends on a direct N domain interaction.

Structural determinants for protein unfolding and translocation by the Hsp104 protein disaggregase

“…Mol 3 matches the F subunit of the casein-bound, closed structure (PDB: 5VY9-F) [ 19 ]. It is noteworthy that the N domain conformation of the Hsp104 1-360 monomer in the hexagonal crystal form (mol 4) [ 25 ] differs from the other three conformations presented here and matches the N domain conformation of the D subunit of the casein-bound, closed structure (PDB: 5VY9-D) [ 19 ]. Together, these findings indicate that the high en bloc mobility of the N domain observed in our crystal structure is likely to be of functional importance, and is also observed in physiologically relevant structures of Hsp104 hexamers.…”

“…The atomic structures of the N and AAA-1 large domains alone are nearly identical amongst the three Hsp104 1-360 molecules and superimpose pairwise with an RMSD of only 0.41 ± 0.05 Å (N domain) and 0.72 ± 0.22 Å (AAA-1 large ). In addition, the three Hsp104 1-360 molecules superimpose pairwise with the hexagonal crystal structure of one Hsp104 1-360 monomer (PDB: 6AMN) [ 25 ] with an RMSD of 0.47 ± 0.01 Å (N domain) and 0.75 ± 0.01 Å (AAA-1 large ), and with the crystal structure of the isolated S. cerevisiae Hsp104 N domain (PDB: 5U2U) [ 34 ] with an RMSD of 0.50 ± 0.05Å calculated over all atoms. Superimposing the crystal structures of the complete Hsp104 1-360 fragment through their AAA-1 large domain shows that the orientation of the N domain seen in mol 1 and mol 2 is rotated by 172–174° relative to that in mol 3 ( Figure 1 ) with residues 161–165 making up the hinge region.…”

“…Data were collected and processed using the HKL2000 software package [ 23 ] (Supplementary Table S1). The crystal structure of Hsp104 1-360 was determined by molecular replacement using MOLREP [ 24 ] with Protein Data Bank (PDB) accession 6AMN as search model [ 25 ]. Two molecules of Hsp104 1-360 were found.…”

Cryo-EM Structures of the Hsp104 Protein Disaggregase Captured in the ATP Conformation

“…Yeast Hsp104 and its bacterial homolog ClpB are ring-forming, ATP-driven protein disaggre-gases that, together with Hsp70 and Hsp40 chaperones, recover functional protein from both amorphous aggregates and amyloid-forming prions ( Doyle et al, 2013 ; Mogk et al, 2015 ). Hsp104 possesses an N-terminal domain (NTD) and two nucleotide-binding domains (NBD1 and NBD2), which facilitate substrate binding ( Gates et al, 2017 ; Hung and Masison, 2006 ; Lee et al, 2017b ) and abolish Hsp104 function when mutated ( Lee et al, 2017a ; Lum et al, 2004 ). A hallmark of Hsp104/ClpB members is the coiled-coil motif within NBD1, which binds Hsp70 ( Miot et al, 2011 ; Rosenzweig et al, 2013 ; Sielaff and Tsai, 2010 ) and, in turn, activates the Hsp104 motor for protein disaggregation ( Lee et al, 2013 ; Seyffer et al, 2012 ).…”

Forms and abundance of chaperone proteins influence yeast prion variant competition

“…Overexpression of Hsp104∆N cured [ PSI + ] less efficiently, hence still permitting the propagation of VK and VL. The caveat is that the mutant exhibited other functional deficiencies, so it might not be an apt proxy for the full‐length protein (Hung and Masison, ; Sweeny et al ., ; Lee et al ., ). We found that Hsp104∆N overexpression in wild‐type cells did not change variant dominance (Fig.…”