Characterization of interactions between the anti-apoptotic protein BAG-1 and Hsc70 molecular chaperones.
The anti-cell death protein BAG-1 binds to 70-kDa heat shock proteins (Hsp70/Hsc70) and modulates their chaperone activity. Among other facilitory roles, BAG-1 may serve as a nucleotide exchange factor for Hsp70/Hsc70 family proteins and thus represents the first example of a eukaryotic homologue of the bacterial co-chaperone GrpE. In this study, the interactions between BAG-1 and Hsc70 are characterized and compared with the analogous GrpE-DnaK bacterial system. In contrast to GrpE, which binds DnaK as a dimer, BAG-1 binds to Hsc70 as a monomer with a 1:1 stoichiometry. Dynamic light scattering, sedimentation equilibrium, and circular dichroism measurements provided evidence that BAG-1 exists as an elongated, highly helical monomer in solution. Isothermal titration microcalorimetry was used to determine the complex stoichiometry and an equilibrium dissociation constant, KD, of 100 nM. Kinetic analysis using surface plasmon resonance yielded a KD consistent with the calorimetrically determined value. Molecular modeling permitted a comparison of structural features between the functionally homologous BAG-1 and GrpE proteins. These data were used to propose a mechanism for BAG-1 in the regulation of Hsp70/Hsc70 chaperone activity.
Stuart
J K
JK
Burnham Institute, Cancer Research Center, La Jolla, California 92037, USA.
Myszka
D G
DG
Joss
L
L
Mitchell
R S
RS
McDonald
S M
SM
Xie
Z
Z
Takayama
S
S
Reed
J C
JC
Ely
K R
KR
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002151:Calorimetry; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002942:Circular Dichroism; D004268:DNA-Binding Proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D014157:Transcription Factors
C091732:BCL2-associated athanogene 1 protein; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C068113:GrpE protein, Bacteria; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D006360:Heat-Shock Proteins; C494372:Hspa8 protein, mouse; D018832:Molecular Chaperones; D012996:Solutions; D014157:Transcription Factors