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Atypical AAA+ subunit packing creates an expanded cavity for disaggregation by the protein-remodeling factor Hsp104.

Wendler P, Shorter J, Plisson C, Cashikar AG, Lindquist S, Saibil HR - Cell (2007)

Bottom Line: This packing results in a greatly expanded cavity, which is capped at either end by N- and C-terminal domains.The fitted structures as well as mutation of conserved coiled-coil arginines suggest that the coiled-coil domain plays a major role in the extraction of proteins from aggregates, providing conserved residues for key functions in ATP hydrolysis and potentially for substrate interaction.The large cavity could enable the uptake of polypeptide loops without a requirement for exposed N or C termini.

View Article: PubMed Central - PubMed

Affiliation: Department of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, UK.

ABSTRACT
Hsp104, a yeast protein-remodeling factor of the AAA+ (ATPases associated with various cellular activities) superfamily, and its homologs in bacteria and plants mediate cell recovery after severe stress by disaggregating denatured proteins through a poorly understood mechanism. Here, we present cryo-electron microscopy maps and domain fitting of Hsp104 hexamers, revealing an unusual arrangement of AAA+ modules with the prominent coiled-coil domain intercalated between the AAA+ domains. This packing results in a greatly expanded cavity, which is capped at either end by N- and C-terminal domains. The fitted structures as well as mutation of conserved coiled-coil arginines suggest that the coiled-coil domain plays a major role in the extraction of proteins from aggregates, providing conserved residues for key functions in ATP hydrolysis and potentially for substrate interaction. The large cavity could enable the uptake of polypeptide loops without a requirement for exposed N or C termini.

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Cryo-EM Images and 3D Reconstructions of Hsp104(A and B) Cryo-EM raw images of Hsp104N728A (A) and Hsp104 ΔN (B) in the presence of ATPγS. Class averages containing 6–16 images of the final dataset, obtained by multivariate statistical analysis in IMAGIC, show characteristic views of the complexes (inset, upper row). Reprojections of the 3D structure in the Euler angle directions assigned to the class averages are included to judge the reconstruction (inset, lower row). The arrows indicate characteristic side views on the raw images.(C–F) Three-dimensional reconstruction of Hsp104N728A and Hsp104 ΔN at 13 Å and 11 Å resolution as side views (C and D) and cut open side views (E and F). N, NBD1, and NBD2 indicate the assignment for the Hsp104 domains. Surface views show the density rendered at a threshold accounting for a molecular mass of 612 kDa (Hsp104N728A) and 500 kDa (Hsp104 ΔN).
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fig1: Cryo-EM Images and 3D Reconstructions of Hsp104(A and B) Cryo-EM raw images of Hsp104N728A (A) and Hsp104 ΔN (B) in the presence of ATPγS. Class averages containing 6–16 images of the final dataset, obtained by multivariate statistical analysis in IMAGIC, show characteristic views of the complexes (inset, upper row). Reprojections of the 3D structure in the Euler angle directions assigned to the class averages are included to judge the reconstruction (inset, lower row). The arrows indicate characteristic side views on the raw images.(C–F) Three-dimensional reconstruction of Hsp104N728A and Hsp104 ΔN at 13 Å and 11 Å resolution as side views (C and D) and cut open side views (E and F). N, NBD1, and NBD2 indicate the assignment for the Hsp104 domains. Surface views show the density rendered at a threshold accounting for a molecular mass of 612 kDa (Hsp104N728A) and 500 kDa (Hsp104 ΔN).

Mentions: Cryo-EM studies on Hsp100 proteins have so far not revealed any clear density for the N-terminal domains, suggesting that these domains are highly mobile in Hsp100 proteins and undergo movements of at least 30 Å (Lee et al., 2003; Ishikawa et al., 2004). To examine the layout of Hsp104, we compared the hexameric structures of full-length Hsp104 (Hsp104N728A) with an N-terminal deletion mutant (Hsp104 ΔN) (Figure 1). All data were collected in presence of the ATP analog ATPγS to promote a stable oligomeric assembly. The sensor-1 mutation in NBD2 has no effect on nucleotide binding but reduces ATP hydrolysis by Hsp104 at low protein and ATP concentrations, and elicits several protein-remodeling activities (Hattendorf and Lindquist, 2002b; Doyle et al., 2007). Analysis of Hsp104 ΔN top views revealed 6-fold symmetry (see Figure S1A available online) and this was used for single particle reconstruction of both maps. Refinement of the maps yielded structures at 11 and 13 Å resolution for Hsp104 ΔN and Hsp104N728A respectively.


Atypical AAA+ subunit packing creates an expanded cavity for disaggregation by the protein-remodeling factor Hsp104.

Wendler P, Shorter J, Plisson C, Cashikar AG, Lindquist S, Saibil HR - Cell (2007)

Cryo-EM Images and 3D Reconstructions of Hsp104(A and B) Cryo-EM raw images of Hsp104N728A (A) and Hsp104 ΔN (B) in the presence of ATPγS. Class averages containing 6–16 images of the final dataset, obtained by multivariate statistical analysis in IMAGIC, show characteristic views of the complexes (inset, upper row). Reprojections of the 3D structure in the Euler angle directions assigned to the class averages are included to judge the reconstruction (inset, lower row). The arrows indicate characteristic side views on the raw images.(C–F) Three-dimensional reconstruction of Hsp104N728A and Hsp104 ΔN at 13 Å and 11 Å resolution as side views (C and D) and cut open side views (E and F). N, NBD1, and NBD2 indicate the assignment for the Hsp104 domains. Surface views show the density rendered at a threshold accounting for a molecular mass of 612 kDa (Hsp104N728A) and 500 kDa (Hsp104 ΔN).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211523&req=5

fig1: Cryo-EM Images and 3D Reconstructions of Hsp104(A and B) Cryo-EM raw images of Hsp104N728A (A) and Hsp104 ΔN (B) in the presence of ATPγS. Class averages containing 6–16 images of the final dataset, obtained by multivariate statistical analysis in IMAGIC, show characteristic views of the complexes (inset, upper row). Reprojections of the 3D structure in the Euler angle directions assigned to the class averages are included to judge the reconstruction (inset, lower row). The arrows indicate characteristic side views on the raw images.(C–F) Three-dimensional reconstruction of Hsp104N728A and Hsp104 ΔN at 13 Å and 11 Å resolution as side views (C and D) and cut open side views (E and F). N, NBD1, and NBD2 indicate the assignment for the Hsp104 domains. Surface views show the density rendered at a threshold accounting for a molecular mass of 612 kDa (Hsp104N728A) and 500 kDa (Hsp104 ΔN).
Mentions: Cryo-EM studies on Hsp100 proteins have so far not revealed any clear density for the N-terminal domains, suggesting that these domains are highly mobile in Hsp100 proteins and undergo movements of at least 30 Å (Lee et al., 2003; Ishikawa et al., 2004). To examine the layout of Hsp104, we compared the hexameric structures of full-length Hsp104 (Hsp104N728A) with an N-terminal deletion mutant (Hsp104 ΔN) (Figure 1). All data were collected in presence of the ATP analog ATPγS to promote a stable oligomeric assembly. The sensor-1 mutation in NBD2 has no effect on nucleotide binding but reduces ATP hydrolysis by Hsp104 at low protein and ATP concentrations, and elicits several protein-remodeling activities (Hattendorf and Lindquist, 2002b; Doyle et al., 2007). Analysis of Hsp104 ΔN top views revealed 6-fold symmetry (see Figure S1A available online) and this was used for single particle reconstruction of both maps. Refinement of the maps yielded structures at 11 and 13 Å resolution for Hsp104 ΔN and Hsp104N728A respectively.

Bottom Line: This packing results in a greatly expanded cavity, which is capped at either end by N- and C-terminal domains.The fitted structures as well as mutation of conserved coiled-coil arginines suggest that the coiled-coil domain plays a major role in the extraction of proteins from aggregates, providing conserved residues for key functions in ATP hydrolysis and potentially for substrate interaction.The large cavity could enable the uptake of polypeptide loops without a requirement for exposed N or C termini.

View Article: PubMed Central - PubMed

Affiliation: Department of Crystallography, Birkbeck College, Malet Street, London WC1E 7HX, UK.

ABSTRACT
Hsp104, a yeast protein-remodeling factor of the AAA+ (ATPases associated with various cellular activities) superfamily, and its homologs in bacteria and plants mediate cell recovery after severe stress by disaggregating denatured proteins through a poorly understood mechanism. Here, we present cryo-electron microscopy maps and domain fitting of Hsp104 hexamers, revealing an unusual arrangement of AAA+ modules with the prominent coiled-coil domain intercalated between the AAA+ domains. This packing results in a greatly expanded cavity, which is capped at either end by N- and C-terminal domains. The fitted structures as well as mutation of conserved coiled-coil arginines suggest that the coiled-coil domain plays a major role in the extraction of proteins from aggregates, providing conserved residues for key functions in ATP hydrolysis and potentially for substrate interaction. The large cavity could enable the uptake of polypeptide loops without a requirement for exposed N or C termini.

Show MeSH