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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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Comparison of Subunits from the ClpB and ClpA Crystal Structures with the Hsp104 Hexamer ModelComparison of subunits from the ClpB (1QVR_A, Lee et al., 2003) and ClpA (1R6B, Xia et al., 2004) crystal structures with the Hsp104 hexamer model.(A) Front view of the three structures aligned by superimposition of the Cα's in NBD1. The position of Hsp104 NBD1 along the rotational axis of the Hsp104 hexamer is indicated in red. Color code is as in Figure 2.(B) Same as in (A), but with 120° anticlockwise rotation of the monomers.(C) Hexameric assembly of Hsp104 homology model derived from the fit into the cryo EM maps.(D) Cartoon of subunit arrangement in Hsp104 oligomer. NBD and N domains are labeled and color coded as in (A). The length of the flexible hinge between N and NBD1 allows either N or N' to connect to NBD1.
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fig3: Comparison of Subunits from the ClpB and ClpA Crystal Structures with the Hsp104 Hexamer ModelComparison of subunits from the ClpB (1QVR_A, Lee et al., 2003) and ClpA (1R6B, Xia et al., 2004) crystal structures with the Hsp104 hexamer model.(A) Front view of the three structures aligned by superimposition of the Cα's in NBD1. The position of Hsp104 NBD1 along the rotational axis of the Hsp104 hexamer is indicated in red. Color code is as in Figure 2.(B) Same as in (A), but with 120° anticlockwise rotation of the monomers.(C) Hexameric assembly of Hsp104 homology model derived from the fit into the cryo EM maps.(D) Cartoon of subunit arrangement in Hsp104 oligomer. NBD and N domains are labeled and color coded as in (A). The length of the flexible hinge between N and NBD1 allows either N or N' to connect to NBD1.

Mentions: When the monomeric Hsp100 structures from the crystal and the EM fits are superimposed through the Cα atoms of NBD1, the most significant difference is the position of the coiled-coil domain (Figure 3). In the EM conformation, it is located on the inside of NBD1, forming a close contact with this domain, whereas the crystal conformation positions it on the outside without major contacts to the rest of the molecule (Figure 3A). Nevertheless, when viewed from the side (Figure 3B), the relative positions of the coiled coil and NBD1 are similar. Thus, the EM conformation can be generated from the crystal conformation by rotating the coiled coil ∼90° clockwise around helix L2 and ∼90° anticlockwise around the hinge connection to NBD1. The orientation of NBD2 and the N-terminal domain in the fit to the EM map differ from those of ClpB and ClpA subunit crystal structures, emphasizing the mobility around the hinge points. In our structure, the coiled coil contacts an N domain at the L3/L4 end, interacts extensively with NBD1 of the same subunit and contacts NBD2 of the adjacent subunit through the L1/L2 end, providing a first glimpse of the structural basis for the complex allosteric interactions in this protein family (Figures 3C and 3D).


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)

Comparison of Subunits from the ClpB and ClpA Crystal Structures with the Hsp104 Hexamer ModelComparison of subunits from the ClpB (1QVR_A, Lee et al., 2003) and ClpA (1R6B, Xia et al., 2004) crystal structures with the Hsp104 hexamer model.(A) Front view of the three structures aligned by superimposition of the Cα's in NBD1. The position of Hsp104 NBD1 along the rotational axis of the Hsp104 hexamer is indicated in red. Color code is as in Figure 2.(B) Same as in (A), but with 120° anticlockwise rotation of the monomers.(C) Hexameric assembly of Hsp104 homology model derived from the fit into the cryo EM maps.(D) Cartoon of subunit arrangement in Hsp104 oligomer. NBD and N domains are labeled and color coded as in (A). The length of the flexible hinge between N and NBD1 allows either N or N' to connect to NBD1.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Comparison of Subunits from the ClpB and ClpA Crystal Structures with the Hsp104 Hexamer ModelComparison of subunits from the ClpB (1QVR_A, Lee et al., 2003) and ClpA (1R6B, Xia et al., 2004) crystal structures with the Hsp104 hexamer model.(A) Front view of the three structures aligned by superimposition of the Cα's in NBD1. The position of Hsp104 NBD1 along the rotational axis of the Hsp104 hexamer is indicated in red. Color code is as in Figure 2.(B) Same as in (A), but with 120° anticlockwise rotation of the monomers.(C) Hexameric assembly of Hsp104 homology model derived from the fit into the cryo EM maps.(D) Cartoon of subunit arrangement in Hsp104 oligomer. NBD and N domains are labeled and color coded as in (A). The length of the flexible hinge between N and NBD1 allows either N or N' to connect to NBD1.
Mentions: When the monomeric Hsp100 structures from the crystal and the EM fits are superimposed through the Cα atoms of NBD1, the most significant difference is the position of the coiled-coil domain (Figure 3). In the EM conformation, it is located on the inside of NBD1, forming a close contact with this domain, whereas the crystal conformation positions it on the outside without major contacts to the rest of the molecule (Figure 3A). Nevertheless, when viewed from the side (Figure 3B), the relative positions of the coiled coil and NBD1 are similar. Thus, the EM conformation can be generated from the crystal conformation by rotating the coiled coil ∼90° clockwise around helix L2 and ∼90° anticlockwise around the hinge connection to NBD1. The orientation of NBD2 and the N-terminal domain in the fit to the EM map differ from those of ClpB and ClpA subunit crystal structures, emphasizing the mobility around the hinge points. In our structure, the coiled coil contacts an N domain at the L3/L4 end, interacts extensively with NBD1 of the same subunit and contacts NBD2 of the adjacent subunit through the L1/L2 end, providing a first glimpse of the structural basis for the complex allosteric interactions in this protein family (Figures 3C and 3D).

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