The carboxyl-terminal lobe of Hsc70 ATPase domain is sufficient for binding to BAG1

Lars Brive; Shinichi Takayama; Klára Briknarová; Sachiko Homma; Stacie K. Ishida; John C. Reed; Kathryn R. Ely

doi:10.1006/bbrc.2001.6087

The carboxyl-terminal lobe of Hsc70 ATPase domain is sufficient for binding to BAG1

Lars Brive
, Shinichi Takayama
, Klára Briknarová
, Sachiko Homma
, Stacie K. Ishida
, John C. Reed
, Kathryn R. Ely

Chemistry and Biochemistry

Sanford Burnham Prebys Medical Discovery Institute

Research output: Contribution to journal › Article › peer-review

48 Scopus citations

Abstract

The molecular co-chaperone BAG1 and other members of the BAG family bind to Hsp70/Hsc70 heat shock proteins through a conserved BAG domain that interacts with the ATPase domain of the chaperone. BAG1 and other accessory proteins stimulate ATP hydrolysis and regulate the ATP-driven activity of the chaperone complexes. Contacts are made through residues in helices α2 and α3 of the BAG domain and predominately residues in the C-terminal lobe of the bi-lobed Hsc70 ATPase domain. Within the C-terminal lobe, a subdomain exists that contains all the contacts shown by mutagenesis to be required for BAG1 recognition. In this study, the subdomain, representing Hsc70 residues 229-309, was cloned and expressed as a separately folded unit. The results of in vitro binding assays demonstrate that this subdomain is sufficient for binding to BAG1. Binding analyses with surface plasmon resonance indicated that the subdomain binds to BAG1 with a 10-fold decrease in equilibrium dissociation constant (K_D = 22 nM) relative to the intact ATPase domain. This result suggests that the stabilizing contacts for docking of BAG1 to Hsc70 are located in the C-terminal lobe of the ATPase domain. These findings provide new insights into the role of co-chaperones as nucleotide exchange factors.

Original language	English
Pages (from-to)	1099-1105
Number of pages	7
Journal	Biochemical and Biophysical Research Communications
Volume	289
Issue number	5
DOIs	https://doi.org/10.1006/bbrc.2001.6087
State	Published - Dec 21 2001

Funding

The authors are grateful to M. Havert for purification of the BAG1 protein, N. Preece for assistance with the NMR spectrometer, C. Lombardo for advice with the surface plasmon resonance experiments, and S. Hammond for preparation of the manuscript for publication. This study was funded by grants from the Human Frontier Science Program (to L.B.), The California Breast Cancer Research Program (to K.R.E.), CaP CURE (to K.R.E.), the National Institutes of Health (to J.C.R.), the State of California Cancer Research Program (to S.T.) and the USAMRDC Breast Cancer Research Program (to S.T.).

Keywords

Apoptosis
BAG family
Heat shock protein
Molecular chaperones
Nucleotide exchange factor

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10.1006/bbrc.2001.6087

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title = "The carboxyl-terminal lobe of Hsc70 ATPase domain is sufficient for binding to BAG1",

abstract = "The molecular co-chaperone BAG1 and other members of the BAG family bind to Hsp70/Hsc70 heat shock proteins through a conserved BAG domain that interacts with the ATPase domain of the chaperone. BAG1 and other accessory proteins stimulate ATP hydrolysis and regulate the ATP-driven activity of the chaperone complexes. Contacts are made through residues in helices α2 and α3 of the BAG domain and predominately residues in the C-terminal lobe of the bi-lobed Hsc70 ATPase domain. Within the C-terminal lobe, a subdomain exists that contains all the contacts shown by mutagenesis to be required for BAG1 recognition. In this study, the subdomain, representing Hsc70 residues 229-309, was cloned and expressed as a separately folded unit. The results of in vitro binding assays demonstrate that this subdomain is sufficient for binding to BAG1. Binding analyses with surface plasmon resonance indicated that the subdomain binds to BAG1 with a 10-fold decrease in equilibrium dissociation constant (KD = 22 nM) relative to the intact ATPase domain. This result suggests that the stabilizing contacts for docking of BAG1 to Hsc70 are located in the C-terminal lobe of the ATPase domain. These findings provide new insights into the role of co-chaperones as nucleotide exchange factors.",

keywords = "Apoptosis, BAG family, Heat shock protein, Molecular chaperones, Nucleotide exchange factor",

author = "Lars Brive and Shinichi Takayama and Kl{\'a}ra Briknarov{\'a} and Sachiko Homma and Ishida, \{Stacie K.\} and Reed, \{John C.\} and Ely, \{Kathryn R.\}",

note = "Funding Information: The authors are grateful to M. Havert for purification of the BAG1 protein, N. Preece for assistance with the NMR spectrometer, C. Lombardo for advice with the surface plasmon resonance experiments, and S. Hammond for preparation of the manuscript for publication. This study was funded by grants from the Human Frontier Science Program (to L.B.), The California Breast Cancer Research Program (to K.R.E.), CaP CURE (to K.R.E.), the National Institutes of Health (to J.C.R.), the State of California Cancer Research Program (to S.T.) and the USAMRDC Breast Cancer Research Program (to S.T.).",

year = "2001",

month = dec,

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doi = "10.1006/bbrc.2001.6087",

language = "English",

volume = "289",

pages = "1099--1105",

journal = "Biochemical and Biophysical Research Communications",

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TY - JOUR

T1 - The carboxyl-terminal lobe of Hsc70 ATPase domain is sufficient for binding to BAG1

AU - Brive, Lars

AU - Takayama, Shinichi

AU - Briknarová, Klára

AU - Homma, Sachiko

AU - Ishida, Stacie K.

AU - Reed, John C.

AU - Ely, Kathryn R.

N1 - Funding Information: The authors are grateful to M. Havert for purification of the BAG1 protein, N. Preece for assistance with the NMR spectrometer, C. Lombardo for advice with the surface plasmon resonance experiments, and S. Hammond for preparation of the manuscript for publication. This study was funded by grants from the Human Frontier Science Program (to L.B.), The California Breast Cancer Research Program (to K.R.E.), CaP CURE (to K.R.E.), the National Institutes of Health (to J.C.R.), the State of California Cancer Research Program (to S.T.) and the USAMRDC Breast Cancer Research Program (to S.T.).

PY - 2001/12/21

Y1 - 2001/12/21

N2 - The molecular co-chaperone BAG1 and other members of the BAG family bind to Hsp70/Hsc70 heat shock proteins through a conserved BAG domain that interacts with the ATPase domain of the chaperone. BAG1 and other accessory proteins stimulate ATP hydrolysis and regulate the ATP-driven activity of the chaperone complexes. Contacts are made through residues in helices α2 and α3 of the BAG domain and predominately residues in the C-terminal lobe of the bi-lobed Hsc70 ATPase domain. Within the C-terminal lobe, a subdomain exists that contains all the contacts shown by mutagenesis to be required for BAG1 recognition. In this study, the subdomain, representing Hsc70 residues 229-309, was cloned and expressed as a separately folded unit. The results of in vitro binding assays demonstrate that this subdomain is sufficient for binding to BAG1. Binding analyses with surface plasmon resonance indicated that the subdomain binds to BAG1 with a 10-fold decrease in equilibrium dissociation constant (KD = 22 nM) relative to the intact ATPase domain. This result suggests that the stabilizing contacts for docking of BAG1 to Hsc70 are located in the C-terminal lobe of the ATPase domain. These findings provide new insights into the role of co-chaperones as nucleotide exchange factors.

AB - The molecular co-chaperone BAG1 and other members of the BAG family bind to Hsp70/Hsc70 heat shock proteins through a conserved BAG domain that interacts with the ATPase domain of the chaperone. BAG1 and other accessory proteins stimulate ATP hydrolysis and regulate the ATP-driven activity of the chaperone complexes. Contacts are made through residues in helices α2 and α3 of the BAG domain and predominately residues in the C-terminal lobe of the bi-lobed Hsc70 ATPase domain. Within the C-terminal lobe, a subdomain exists that contains all the contacts shown by mutagenesis to be required for BAG1 recognition. In this study, the subdomain, representing Hsc70 residues 229-309, was cloned and expressed as a separately folded unit. The results of in vitro binding assays demonstrate that this subdomain is sufficient for binding to BAG1. Binding analyses with surface plasmon resonance indicated that the subdomain binds to BAG1 with a 10-fold decrease in equilibrium dissociation constant (KD = 22 nM) relative to the intact ATPase domain. This result suggests that the stabilizing contacts for docking of BAG1 to Hsc70 are located in the C-terminal lobe of the ATPase domain. These findings provide new insights into the role of co-chaperones as nucleotide exchange factors.

KW - Apoptosis

KW - BAG family

KW - Heat shock protein

KW - Molecular chaperones

KW - Nucleotide exchange factor

UR - https://www.scopus.com/pages/publications/0035931003

U2 - 10.1006/bbrc.2001.6087

DO - 10.1006/bbrc.2001.6087

M3 - Article

C2 - 11741305

AN - SCOPUS:0035931003

SN - 0006-291X

VL - 289

SP - 1099

EP - 1105

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

IS - 5

ER -

The carboxyl-terminal lobe of Hsc70 ATPase domain is sufficient for binding to BAG1

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Keywords

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