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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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Positions of Key Residues in the Hsp104 Hexamer ModelTwo Hsp104 monomers, colored in dark and light blue, are fitted into the EM map. The coiled-coil domain is colored green in both monomers. Conserved arginine residues R419, R444, and R495 are shown as red spheres. Predicted arginine fingers R334 and R765 are shown as red sticks. L462R and D794N mutations are shown as magenta spheres. ATP is shown in ball and stick representation.
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fig5: Positions of Key Residues in the Hsp104 Hexamer ModelTwo Hsp104 monomers, colored in dark and light blue, are fitted into the EM map. The coiled-coil domain is colored green in both monomers. Conserved arginine residues R419, R444, and R495 are shown as red spheres. Predicted arginine fingers R334 and R765 are shown as red sticks. L462R and D794N mutations are shown as magenta spheres. ATP is shown in ball and stick representation.

Mentions: A sequence alignment of 26 Hsp100 proteins revealed the presence of three highly conserved arginine residues within the coiled-coil domain: R419, R444, and R495 (Figure S4). Arginine 419 is located on Helix L1 of the coiled coil and in our model could potentially act as an arginine finger for NBD1. Arginine 495 and R444 are positioned at opposite ends of Helix L2 with R444 contacting NBD2 of the neighboring subunit (Figure 5). To investigate potential roles of these arginine residues in ATP hydrolysis we mutated each to methionine and compared them with effects of mutating the predicted arginine fingers R334 (NBD1) and R765 (NBD2) (Neuwald et al., 1999).


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)

Positions of Key Residues in the Hsp104 Hexamer ModelTwo Hsp104 monomers, colored in dark and light blue, are fitted into the EM map. The coiled-coil domain is colored green in both monomers. Conserved arginine residues R419, R444, and R495 are shown as red spheres. Predicted arginine fingers R334 and R765 are shown as red sticks. L462R and D794N mutations are shown as magenta spheres. ATP is shown in ball and stick representation.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Positions of Key Residues in the Hsp104 Hexamer ModelTwo Hsp104 monomers, colored in dark and light blue, are fitted into the EM map. The coiled-coil domain is colored green in both monomers. Conserved arginine residues R419, R444, and R495 are shown as red spheres. Predicted arginine fingers R334 and R765 are shown as red sticks. L462R and D794N mutations are shown as magenta spheres. ATP is shown in ball and stick representation.
Mentions: A sequence alignment of 26 Hsp100 proteins revealed the presence of three highly conserved arginine residues within the coiled-coil domain: R419, R444, and R495 (Figure S4). Arginine 419 is located on Helix L1 of the coiled coil and in our model could potentially act as an arginine finger for NBD1. Arginine 495 and R444 are positioned at opposite ends of Helix L2 with R444 contacting NBD2 of the neighboring subunit (Figure 5). To investigate potential roles of these arginine residues in ATP hydrolysis we mutated each to methionine and compared them with effects of mutating the predicted arginine fingers R334 (NBD1) and R765 (NBD2) (Neuwald et al., 1999).

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