Structural analysis of BAG1 cochaperone and its interactions with Hsc70 heat shock protein

BAG-family proteins share a conserved protein interaction region, called th e 'BAG domain' which binds and regulates Hsp70/Hsc70 molecular chaperones. This family of cochaperones functionally regulates signal transducing prote ins and transcription factors important for cell stress responses, apoptosi s, proliferation, cell migration and hormone action. Aberrant overexpressio n of the founding member of this family, BAG1, occurs in human cancers. In this study, a structure-based approach was used to identify interacting res idues in a BAG1-Hsc70 complex. An Hsc70-binding fragment of BAG1 was shown by multidimensional NMR methods to consist of an antiparallel three-helix b undle. NMR chemical shift experiments marked surface residues on the second (alpha2) and third (alpha3) helices in the BAG domain that are involved in chaperone binding. Structural predictions were confirmed by site-directed mutagenesis of these residues, resulting in loss of binding of BAG1 to Hsc7 0 in vitro and in cells. Molecular docking of BAG1 to Hsc70 and mutagenesis of Hsc70 marked the molecular surface of the ATPase domain necessary for i nteraction with BAG1. The results provide a structural basis for understand ing the mechanism by which BAG proteins link molecular chaperones and cell signaling pathways.