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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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Biochemical and Functional Consequences of Mutating Conserved Arginine 334, 419, 444, 495, or 765 in Hsp104(A) Wild-type or mutant Hsp104 proteins (0.2 μM) were incubated for 0–20 min at 25°C with ATP (1 mM). At various times the amount of ATP hydrolysis was determined. Values represent the mean ± SD (n = 5).(B) Wild-type (wt) or mutant Hsp104 proteins (0.2 μM) in the presence of ATP (1 mM) were either crosslinked with 0.1% glutaraldehyde for 10 min or left untreated.(C) NM fibers (2.5 μM monomer) were incubated with wild-type or the indicated mutant Hsp104 (2 μM) plus ATP (10 mM) for 60 min at 25°C. Disassembly was monitored by thioflavin T (ThT) fluorescence. Values represent the mean ± SD (n = 4).(D–G) Wild-type (D and F) or Δhsp104 (E and G) cells harboring the indicated plasmid were grown to mid-log phase (5 × 106 cells/ml) in SD-ura liquid. Prior to the 50°C heat treatment, matched cultures were either maintained at 30°C (D and E) or preincubated at 37°C for 30 min (F and G). Following treatment at 50°C for 0–20 min cells were transferred to ice, diluted in ice-cold SD-ura, and immediately plated on SD-ura. Values represent the mean ± SD (n = 3).
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fig6: Biochemical and Functional Consequences of Mutating Conserved Arginine 334, 419, 444, 495, or 765 in Hsp104(A) Wild-type or mutant Hsp104 proteins (0.2 μM) were incubated for 0–20 min at 25°C with ATP (1 mM). At various times the amount of ATP hydrolysis was determined. Values represent the mean ± SD (n = 5).(B) Wild-type (wt) or mutant Hsp104 proteins (0.2 μM) in the presence of ATP (1 mM) were either crosslinked with 0.1% glutaraldehyde for 10 min or left untreated.(C) NM fibers (2.5 μM monomer) were incubated with wild-type or the indicated mutant Hsp104 (2 μM) plus ATP (10 mM) for 60 min at 25°C. Disassembly was monitored by thioflavin T (ThT) fluorescence. Values represent the mean ± SD (n = 4).(D–G) Wild-type (D and F) or Δhsp104 (E and G) cells harboring the indicated plasmid were grown to mid-log phase (5 × 106 cells/ml) in SD-ura liquid. Prior to the 50°C heat treatment, matched cultures were either maintained at 30°C (D and E) or preincubated at 37°C for 30 min (F and G). Following treatment at 50°C for 0–20 min cells were transferred to ice, diluted in ice-cold SD-ura, and immediately plated on SD-ura. Values represent the mean ± SD (n = 3).

Mentions: Since the arginine finger catalyzes ATP hydrolysis in the adjacent subunit by stabilizing the developing negative charge of the transition state, we expect ATPase activity to be reduced when this arginine is mutated to the non-polar amino acid methionine. Indeed, the previously predicted NBD1 arginine finger mutant Hsp104R334M as well as the coiled-coil domain mutants Hsp104R419M and Hsp104R444M each reduced the ATPase activity to ∼20%–30% of wild-type after 20 min at 25°C (Figure 6A). The predicted NBD2 arginine finger mutant, Hsp104R765M, initially displayed reduced ATPase activity, but by the end of the reaction ATPase activity approached wild-type levels. Unexpectedly, Hsp104R495M had ∼3-fold higher ATPase activity than wild-type Hsp104, and the initial rate was more than 10-fold higher than wild-type. Thus, all the conserved arginines affected ATP hydrolysis in some manner.


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)

Biochemical and Functional Consequences of Mutating Conserved Arginine 334, 419, 444, 495, or 765 in Hsp104(A) Wild-type or mutant Hsp104 proteins (0.2 μM) were incubated for 0–20 min at 25°C with ATP (1 mM). At various times the amount of ATP hydrolysis was determined. Values represent the mean ± SD (n = 5).(B) Wild-type (wt) or mutant Hsp104 proteins (0.2 μM) in the presence of ATP (1 mM) were either crosslinked with 0.1% glutaraldehyde for 10 min or left untreated.(C) NM fibers (2.5 μM monomer) were incubated with wild-type or the indicated mutant Hsp104 (2 μM) plus ATP (10 mM) for 60 min at 25°C. Disassembly was monitored by thioflavin T (ThT) fluorescence. Values represent the mean ± SD (n = 4).(D–G) Wild-type (D and F) or Δhsp104 (E and G) cells harboring the indicated plasmid were grown to mid-log phase (5 × 106 cells/ml) in SD-ura liquid. Prior to the 50°C heat treatment, matched cultures were either maintained at 30°C (D and E) or preincubated at 37°C for 30 min (F and G). Following treatment at 50°C for 0–20 min cells were transferred to ice, diluted in ice-cold SD-ura, and immediately plated on SD-ura. Values represent the mean ± SD (n = 3).
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Related In: Results  -  Collection

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fig6: Biochemical and Functional Consequences of Mutating Conserved Arginine 334, 419, 444, 495, or 765 in Hsp104(A) Wild-type or mutant Hsp104 proteins (0.2 μM) were incubated for 0–20 min at 25°C with ATP (1 mM). At various times the amount of ATP hydrolysis was determined. Values represent the mean ± SD (n = 5).(B) Wild-type (wt) or mutant Hsp104 proteins (0.2 μM) in the presence of ATP (1 mM) were either crosslinked with 0.1% glutaraldehyde for 10 min or left untreated.(C) NM fibers (2.5 μM monomer) were incubated with wild-type or the indicated mutant Hsp104 (2 μM) plus ATP (10 mM) for 60 min at 25°C. Disassembly was monitored by thioflavin T (ThT) fluorescence. Values represent the mean ± SD (n = 4).(D–G) Wild-type (D and F) or Δhsp104 (E and G) cells harboring the indicated plasmid were grown to mid-log phase (5 × 106 cells/ml) in SD-ura liquid. Prior to the 50°C heat treatment, matched cultures were either maintained at 30°C (D and E) or preincubated at 37°C for 30 min (F and G). Following treatment at 50°C for 0–20 min cells were transferred to ice, diluted in ice-cold SD-ura, and immediately plated on SD-ura. Values represent the mean ± SD (n = 3).
Mentions: Since the arginine finger catalyzes ATP hydrolysis in the adjacent subunit by stabilizing the developing negative charge of the transition state, we expect ATPase activity to be reduced when this arginine is mutated to the non-polar amino acid methionine. Indeed, the previously predicted NBD1 arginine finger mutant Hsp104R334M as well as the coiled-coil domain mutants Hsp104R419M and Hsp104R444M each reduced the ATPase activity to ∼20%–30% of wild-type after 20 min at 25°C (Figure 6A). The predicted NBD2 arginine finger mutant, Hsp104R765M, initially displayed reduced ATPase activity, but by the end of the reaction ATPase activity approached wild-type levels. Unexpectedly, Hsp104R495M had ∼3-fold higher ATPase activity than wild-type Hsp104, and the initial rate was more than 10-fold higher than wild-type. Thus, all the conserved arginines affected ATP hydrolysis in some manner.

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