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Biological and structural basis for Aha1 regulation of Hsp90 ATPase activity in maintaining proteostasis in the human disease cystic fibrosis.

Koulov AV, LaPointe P, Lu B, Razvi A, Coppinger J, Dong MQ, Matteson J, Laister R, Arrowsmith C, Yates JR, Balch WE - Mol. Biol. Cell (2010)

Bottom Line: The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90.We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client.We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (DeltaF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.

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Analysis of binding of Aha1 cross-linked to Hsp90. (A) A 4–12% gradient SDS-PAGE of purified Hsp90 (lane a) and Aha1 (lane b). Full-length (lanes e, h, and k), N-terminal domain (lanes c, f, and i), or C-terminal domain (lanes d, g, and j) were incubated in the presence of Hsp90 (c–h) or alone in the presence of zero-length (EDC) cross-linker (lanes i–l) in the presence of 1× (c–e) or 2× (f–h) cross-linker as described in Materials and Methods. No cross-linking was observed in any incubation that only contained full-length, or N- and C-terminal domains of Aha1. Incubation of Hsp90 with EDC revealed higher order oligomers reflecting its known C-terminal and N-terminal interaction motifs. Black arrowheads show the Aha1–Hsp90 complexes captured by the EDC cross-linker. The position of the Hsp90 is indicated by the asterisk. Migration of indicated molecular weight markers are shown to the left of the panel. (B) Hsp90 peptides cross-linked to Aha1 are colored in yellow (also see Table 2). Aha1 peptide cross-linked to Hsp90 is shown in yellow on the structure of the C-terminal domain of the protein (PBD: 1X53). Protected residues identified by footprinting in the Hsp90 N-terminal and middle domain (see Figure 3) are shown in red; Aha1 C-terminal residues protected from modification in Aha1–Hsp90 complexes (see Figure 3) are shown in orange. (C) Full-length Aha1 was modified with ANB-NOS, incubated with unlabeled Hsp90, and complexes were photocross-linked for 1 min (lane c), 2 min (lane d), and 3 min (lane e) and separated using a 4–12% gradient gel as described in Materials and Methods. Higher order oligomers are not observed upon incubation of unmodified Hsp90 (lane a) alone or cross-linker modified Aha1 (lane b) alone. The potential migration position of an Hsp90 dimer based on molecular weight markers (left) is shown by the asterisk (see A). Higher molecular weight bands >220 kDa represent complexes of Aha1 with two or more monomers of Hsp90.
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Figure 4: Analysis of binding of Aha1 cross-linked to Hsp90. (A) A 4–12% gradient SDS-PAGE of purified Hsp90 (lane a) and Aha1 (lane b). Full-length (lanes e, h, and k), N-terminal domain (lanes c, f, and i), or C-terminal domain (lanes d, g, and j) were incubated in the presence of Hsp90 (c–h) or alone in the presence of zero-length (EDC) cross-linker (lanes i–l) in the presence of 1× (c–e) or 2× (f–h) cross-linker as described in Materials and Methods. No cross-linking was observed in any incubation that only contained full-length, or N- and C-terminal domains of Aha1. Incubation of Hsp90 with EDC revealed higher order oligomers reflecting its known C-terminal and N-terminal interaction motifs. Black arrowheads show the Aha1–Hsp90 complexes captured by the EDC cross-linker. The position of the Hsp90 is indicated by the asterisk. Migration of indicated molecular weight markers are shown to the left of the panel. (B) Hsp90 peptides cross-linked to Aha1 are colored in yellow (also see Table 2). Aha1 peptide cross-linked to Hsp90 is shown in yellow on the structure of the C-terminal domain of the protein (PBD: 1X53). Protected residues identified by footprinting in the Hsp90 N-terminal and middle domain (see Figure 3) are shown in red; Aha1 C-terminal residues protected from modification in Aha1–Hsp90 complexes (see Figure 3) are shown in orange. (C) Full-length Aha1 was modified with ANB-NOS, incubated with unlabeled Hsp90, and complexes were photocross-linked for 1 min (lane c), 2 min (lane d), and 3 min (lane e) and separated using a 4–12% gradient gel as described in Materials and Methods. Higher order oligomers are not observed upon incubation of unmodified Hsp90 (lane a) alone or cross-linker modified Aha1 (lane b) alone. The potential migration position of an Hsp90 dimer based on molecular weight markers (left) is shown by the asterisk (see A). Higher molecular weight bands >220 kDa represent complexes of Aha1 with two or more monomers of Hsp90.

Mentions: To capture the site(s) of protein–protein interactions between Aha1 and Hsp90, we used a stringent, zero-length cross-linker, EDC. This cross-linker forms a peptide bond between side chains of amino-containing amino acids (such as Lys or Arg) and an acidic residue (Glu or an Asp), residing in immediate proximity in the protein complex. Under physiological salt conditions, we detected higher order complexes (∼5% of total Aha1/Hsp90 added) that reflect interaction of Aha1 full-length or each of the C- and N-terminal domains with either Hsp90 monomer and dimers (Figure 4A, lanes c–h, black arrowheads). These were not detected in control incubations containing either purified Hsp90 (Figure 4A, lane l), full-length Aha1 (Figure 4A, lane k), or the individual C- or N-terminal domains in the presence of EDC (Figure 4A, lanes i and j).


Biological and structural basis for Aha1 regulation of Hsp90 ATPase activity in maintaining proteostasis in the human disease cystic fibrosis.

Koulov AV, LaPointe P, Lu B, Razvi A, Coppinger J, Dong MQ, Matteson J, Laister R, Arrowsmith C, Yates JR, Balch WE - Mol. Biol. Cell (2010)

Analysis of binding of Aha1 cross-linked to Hsp90. (A) A 4–12% gradient SDS-PAGE of purified Hsp90 (lane a) and Aha1 (lane b). Full-length (lanes e, h, and k), N-terminal domain (lanes c, f, and i), or C-terminal domain (lanes d, g, and j) were incubated in the presence of Hsp90 (c–h) or alone in the presence of zero-length (EDC) cross-linker (lanes i–l) in the presence of 1× (c–e) or 2× (f–h) cross-linker as described in Materials and Methods. No cross-linking was observed in any incubation that only contained full-length, or N- and C-terminal domains of Aha1. Incubation of Hsp90 with EDC revealed higher order oligomers reflecting its known C-terminal and N-terminal interaction motifs. Black arrowheads show the Aha1–Hsp90 complexes captured by the EDC cross-linker. The position of the Hsp90 is indicated by the asterisk. Migration of indicated molecular weight markers are shown to the left of the panel. (B) Hsp90 peptides cross-linked to Aha1 are colored in yellow (also see Table 2). Aha1 peptide cross-linked to Hsp90 is shown in yellow on the structure of the C-terminal domain of the protein (PBD: 1X53). Protected residues identified by footprinting in the Hsp90 N-terminal and middle domain (see Figure 3) are shown in red; Aha1 C-terminal residues protected from modification in Aha1–Hsp90 complexes (see Figure 3) are shown in orange. (C) Full-length Aha1 was modified with ANB-NOS, incubated with unlabeled Hsp90, and complexes were photocross-linked for 1 min (lane c), 2 min (lane d), and 3 min (lane e) and separated using a 4–12% gradient gel as described in Materials and Methods. Higher order oligomers are not observed upon incubation of unmodified Hsp90 (lane a) alone or cross-linker modified Aha1 (lane b) alone. The potential migration position of an Hsp90 dimer based on molecular weight markers (left) is shown by the asterisk (see A). Higher molecular weight bands >220 kDa represent complexes of Aha1 with two or more monomers of Hsp90.
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Related In: Results  -  Collection

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Figure 4: Analysis of binding of Aha1 cross-linked to Hsp90. (A) A 4–12% gradient SDS-PAGE of purified Hsp90 (lane a) and Aha1 (lane b). Full-length (lanes e, h, and k), N-terminal domain (lanes c, f, and i), or C-terminal domain (lanes d, g, and j) were incubated in the presence of Hsp90 (c–h) or alone in the presence of zero-length (EDC) cross-linker (lanes i–l) in the presence of 1× (c–e) or 2× (f–h) cross-linker as described in Materials and Methods. No cross-linking was observed in any incubation that only contained full-length, or N- and C-terminal domains of Aha1. Incubation of Hsp90 with EDC revealed higher order oligomers reflecting its known C-terminal and N-terminal interaction motifs. Black arrowheads show the Aha1–Hsp90 complexes captured by the EDC cross-linker. The position of the Hsp90 is indicated by the asterisk. Migration of indicated molecular weight markers are shown to the left of the panel. (B) Hsp90 peptides cross-linked to Aha1 are colored in yellow (also see Table 2). Aha1 peptide cross-linked to Hsp90 is shown in yellow on the structure of the C-terminal domain of the protein (PBD: 1X53). Protected residues identified by footprinting in the Hsp90 N-terminal and middle domain (see Figure 3) are shown in red; Aha1 C-terminal residues protected from modification in Aha1–Hsp90 complexes (see Figure 3) are shown in orange. (C) Full-length Aha1 was modified with ANB-NOS, incubated with unlabeled Hsp90, and complexes were photocross-linked for 1 min (lane c), 2 min (lane d), and 3 min (lane e) and separated using a 4–12% gradient gel as described in Materials and Methods. Higher order oligomers are not observed upon incubation of unmodified Hsp90 (lane a) alone or cross-linker modified Aha1 (lane b) alone. The potential migration position of an Hsp90 dimer based on molecular weight markers (left) is shown by the asterisk (see A). Higher molecular weight bands >220 kDa represent complexes of Aha1 with two or more monomers of Hsp90.
Mentions: To capture the site(s) of protein–protein interactions between Aha1 and Hsp90, we used a stringent, zero-length cross-linker, EDC. This cross-linker forms a peptide bond between side chains of amino-containing amino acids (such as Lys or Arg) and an acidic residue (Glu or an Asp), residing in immediate proximity in the protein complex. Under physiological salt conditions, we detected higher order complexes (∼5% of total Aha1/Hsp90 added) that reflect interaction of Aha1 full-length or each of the C- and N-terminal domains with either Hsp90 monomer and dimers (Figure 4A, lanes c–h, black arrowheads). These were not detected in control incubations containing either purified Hsp90 (Figure 4A, lane l), full-length Aha1 (Figure 4A, lane k), or the individual C- or N-terminal domains in the presence of EDC (Figure 4A, lanes i and j).

Bottom Line: The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90.We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client.We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
The activator of Hsp90 ATPase 1, Aha1, has been shown to participate in the Hsp90 chaperone cycle by stimulating the low intrinsic ATPase activity of Hsp90. To elucidate the structural basis for ATPase stimulation of human Hsp90 by human Aha1, we have developed novel mass spectrometry approaches that demonstrate that the N- and C-terminal domains of Aha1 cooperatively bind across the dimer interface of Hsp90 to modulate the ATP hydrolysis cycle and client activity in vivo. Mutations in both the N- and C-terminal domains of Aha1 impair its ability to bind Hsp90 and stimulate its ATPase activity in vitro and impair in vivo the ability of the Hsp90 system to modulate the folding and trafficking of wild-type and variant (DeltaF508) cystic fibrosis transmembrane conductance regulator (CFTR) responsible for the inherited disease cystic fibrosis (CF). We now propose a general model for the role of Aha1 in the Hsp90 ATPase cycle in proteostasis whereby Aha1 regulates the dwell time of Hsp90 with client. We suggest that Aha1 activity integrates chaperone function with client folding energetics by modulating ATPase sensitive N-terminal dimer structural transitions, thereby protecting transient folding intermediates in vivo that could contribute to protein misfolding systems disorders such as CF when destabilized.

Show MeSH
Related in: MedlinePlus