Characterization of Interactions between the Anti-apoptotic Protein BAG-1 and Hsc70 Molecular Chaperones

Joan K Stuart,David G Myszka,Lisa Joss,Richard S Mitchell,Shawn M Mcdonald,Zhihua Xie,Shinichi Takayama,John C Reed,Kathryn R Ely

doi:10.1074/jbc.273.35.22506

Abstract

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.

Highlights

Molecular chaperones assist in protein folding by preventing misfolding and aggregation, but these important molecules play other vital cellular roles such as import, translocation, and degradation of proteins [1,2,3]
The Hsp70 proteins consist of two domains: an amino-terminal ATPase domain (ϳ45 kDa) and a carboxyl-terminal domain that is composed of a 15-kDa substrate-binding module and a 10-kDa module with undefined function
Except in the case of the Surface plasmon resonance (SPR) studies, BAG-1 was cleaved from its fusion partner by thrombin digestion for solution binding experiments

Summary

The abbreviations used are

Hsp70, 70-kDa heat shock protein; BAG-1, Bcl-2-associated athanogene-1; Hsc70, 70-kDa heat shock cognate protein; GST, glutathione S-transferase; GA, glutathione-agarose; SPR, surface plasmon resonance; ITC, isothermal titration calorimetry; RU, resonance unit. The mechanism by which BAG-1 influences the activities of such diverse proteins can probably be attributed to its ability to directly bind Hsp70/ Hsc family proteins, which in turn interact with multiple target proteins in cells. It was proposed recently that BAG-1 may act as a eukaryotic regulator of Hsp activity, with a functional role similar to the action of GrpE on DnaK, the bacterial equivalent of Hsp70 [21]. To test this hypothesis, in the present study we have characterized the binding of BAG-1 to Hsc and have compared the interactions with those reported for GrpE-DnaK complexes. Biophysical analysis and molecular modeling show that the BAG-1 protein shares fewer structural similarities with GrpE than functional ones, suggesting that structural homology may not be necessary for translation into specific function

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION