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 faci litory roles, BAG-1 may serve as a nucleotide exchange factor for Hsp1 0/Hsc70 family proteins and thus represents the first example of a euk aryotic homologue of the bacterial co-chaperone GrpE. In this study, t he interactions between BAG-1 and Hsc70 are characterized and compared with the analogous GrpE-DnaK bacterial system. In contrast to GrpE, w hich 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 exis ts as an elongated, highly helical monomer in solution. Isothermal tit ration microcalorimetry was used to determine the complex stoichiometr y and an equilibrium dissociation constant, K-D, of 100 nM. Kinetic an alysis using surface plasmon resonance yielded a K-D consistent with t he calorimetrically determined value. Molecular modeling permitted a c omparison of structural features between the functionally homologous B AG-1 and GrpE proteins. These data were used to propose a mechanism fo r BAG-1 in the regulation of Hsp70/Hsc70 chaperone activity.