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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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Docking of the Hsp104 Homology Model into the Cryo-EM Maps(A) Rigid body domain fitting into the Hsp104 ΔN map. Domains and subdomains are color coded as follows: NBD1, red/orange; NBD2, blue/cyan; coiled coil, green. The ATP binding pocket is located at the interface between the NBD subdomains (red/orange, blue/cyan). Left: front view of EM map with one monomer fitted. The connection between NBD1 and NBD2 (∗) and density in the NBD2 layer not filled by the docked structure (ellipse) are indicated. Right: 20 Å slices through the side view, in which the fitted hexamer is rotated anticlockwise through a 60° spherical segment.(B) Cross sections through the fitted NBD1 and NBD2 rings of Hsp104 ΔN. Color code is as in (A).(C) Rigid body fit into the Hsp104N728A map. Left: front view with same color code as in (A) and N termini depicted in yellow. Middle: 20 Å slices through the side view. The N-terminal region missing from the Hsp104 homology model is filled in by equivalent ClpB residues and colored in gray. Right: cross-section through the fitted N-terminal ring. Helix L3 in the coiled coil is colored in magenta.
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fig2: Docking of the Hsp104 Homology Model into the Cryo-EM Maps(A) Rigid body domain fitting into the Hsp104 ΔN map. Domains and subdomains are color coded as follows: NBD1, red/orange; NBD2, blue/cyan; coiled coil, green. The ATP binding pocket is located at the interface between the NBD subdomains (red/orange, blue/cyan). Left: front view of EM map with one monomer fitted. The connection between NBD1 and NBD2 (∗) and density in the NBD2 layer not filled by the docked structure (ellipse) are indicated. Right: 20 Å slices through the side view, in which the fitted hexamer is rotated anticlockwise through a 60° spherical segment.(B) Cross sections through the fitted NBD1 and NBD2 rings of Hsp104 ΔN. Color code is as in (A).(C) Rigid body fit into the Hsp104N728A map. Left: front view with same color code as in (A) and N termini depicted in yellow. Middle: 20 Å slices through the side view. The N-terminal region missing from the Hsp104 homology model is filled in by equivalent ClpB residues and colored in gray. Right: cross-section through the fitted N-terminal ring. Helix L3 in the coiled coil is colored in magenta.

Mentions: The assignment of N and NBD layers in both maps is clear from the preceding comparison and the alignment of the ΔN map to the full-length map (see Methods). Next, we determined the hand of the full-length Hsp104 map by tilt experiments (Figure S3). The domain arrangement of the maps is incompatible with the ClpB crystal structure (Lee et al., 2003), so that an Hsp104 homology model was separated into N, NBD1, NBD2 and coiled-coil domains for rigid body fitting. Since the ΔN structure was better resolved, it was used to establish the domain layout (Figure 2A and 2B). Automated fitting of NBD2 places the connection to NBD1 at the interface between the two layers (Figure 2A, ∗). The short hinge region between the AAA+ domains (10 amino acids) requires NBD1 to be placed to the upper right of NBD2. Fitting the boomerang-shaped cross section of NBD1 (Figure 2B) into the round density leaves an empty pocket on the cavity-facing side of NBD1, which accommodates the helix L3-bearing end of the coiled-coil motif well in length and diameter. Consequently, the helix L1/L2-bearing end of the coiled-coil domain passes through the stronger of the two density connections between the AAA+ domains and occupies the unfilled density at the outer top surface of NBD2. Although this fit is not accurate in fine detail and requires some refolding of the linker regions between NBD1 and the coiled coil, it is compatible with all the interdomain connections and clearly provides a good fit to the EM density. The coiled coil follows a similar path in the full-length structure, but it tilts to follow the more outward rotated orientation of NBD1.


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)

Docking of the Hsp104 Homology Model into the Cryo-EM Maps(A) Rigid body domain fitting into the Hsp104 ΔN map. Domains and subdomains are color coded as follows: NBD1, red/orange; NBD2, blue/cyan; coiled coil, green. The ATP binding pocket is located at the interface between the NBD subdomains (red/orange, blue/cyan). Left: front view of EM map with one monomer fitted. The connection between NBD1 and NBD2 (∗) and density in the NBD2 layer not filled by the docked structure (ellipse) are indicated. Right: 20 Å slices through the side view, in which the fitted hexamer is rotated anticlockwise through a 60° spherical segment.(B) Cross sections through the fitted NBD1 and NBD2 rings of Hsp104 ΔN. Color code is as in (A).(C) Rigid body fit into the Hsp104N728A map. Left: front view with same color code as in (A) and N termini depicted in yellow. Middle: 20 Å slices through the side view. The N-terminal region missing from the Hsp104 homology model is filled in by equivalent ClpB residues and colored in gray. Right: cross-section through the fitted N-terminal ring. Helix L3 in the coiled coil is colored in magenta.
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Related In: Results  -  Collection

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fig2: Docking of the Hsp104 Homology Model into the Cryo-EM Maps(A) Rigid body domain fitting into the Hsp104 ΔN map. Domains and subdomains are color coded as follows: NBD1, red/orange; NBD2, blue/cyan; coiled coil, green. The ATP binding pocket is located at the interface between the NBD subdomains (red/orange, blue/cyan). Left: front view of EM map with one monomer fitted. The connection between NBD1 and NBD2 (∗) and density in the NBD2 layer not filled by the docked structure (ellipse) are indicated. Right: 20 Å slices through the side view, in which the fitted hexamer is rotated anticlockwise through a 60° spherical segment.(B) Cross sections through the fitted NBD1 and NBD2 rings of Hsp104 ΔN. Color code is as in (A).(C) Rigid body fit into the Hsp104N728A map. Left: front view with same color code as in (A) and N termini depicted in yellow. Middle: 20 Å slices through the side view. The N-terminal region missing from the Hsp104 homology model is filled in by equivalent ClpB residues and colored in gray. Right: cross-section through the fitted N-terminal ring. Helix L3 in the coiled coil is colored in magenta.
Mentions: The assignment of N and NBD layers in both maps is clear from the preceding comparison and the alignment of the ΔN map to the full-length map (see Methods). Next, we determined the hand of the full-length Hsp104 map by tilt experiments (Figure S3). The domain arrangement of the maps is incompatible with the ClpB crystal structure (Lee et al., 2003), so that an Hsp104 homology model was separated into N, NBD1, NBD2 and coiled-coil domains for rigid body fitting. Since the ΔN structure was better resolved, it was used to establish the domain layout (Figure 2A and 2B). Automated fitting of NBD2 places the connection to NBD1 at the interface between the two layers (Figure 2A, ∗). The short hinge region between the AAA+ domains (10 amino acids) requires NBD1 to be placed to the upper right of NBD2. Fitting the boomerang-shaped cross section of NBD1 (Figure 2B) into the round density leaves an empty pocket on the cavity-facing side of NBD1, which accommodates the helix L3-bearing end of the coiled-coil motif well in length and diameter. Consequently, the helix L1/L2-bearing end of the coiled-coil domain passes through the stronger of the two density connections between the AAA+ domains and occupies the unfilled density at the outer top surface of NBD2. Although this fit is not accurate in fine detail and requires some refolding of the linker regions between NBD1 and the coiled coil, it is compatible with all the interdomain connections and clearly provides a good fit to the EM density. The coiled coil follows a similar path in the full-length structure, but it tilts to follow the more outward rotated orientation of NBD1.

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
Related in: MedlinePlus