Abstract
BAG-family proteins share a conserved protein interaction region, called the 'BAG domain', which binds and regulates Hsp70/Hsc70 molecular chaperones. This family of cochaperones functionally regulates signal transducing proteins and transcription factors important for cell stress responses, apoptosis, proliferation, cell migration and hormone action. Aberrant overexpression of the founding member of this family, BAG1, occurs in human cancers. In this study, a structure-based approach was used to identify interacting residues in a BAG1–Hsc70 complex. An Hsc70-binding fragment of BAG1 was shown by multidimensional NMR methods to consist of an antiparallel three-helix bundle. NMR chemical shift experiments marked surface residues on the second (α2) and third (α3) 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 Hsc70 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 interaction with BAG1. The results provide a structural basis for understanding the mechanism by which BAG proteins link molecular chaperones and cell signaling pathways.
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Acknowledgements
This research was performed in part at the Environmental Molecular Sciences Laboratory (a national scientific user facility sponsored by the U.S. DOE Office of Biological and Environmental Research) located at Pacific Northwest National Laboratory, operated by Battelle for the DOE. The authors are grateful to the staff at the HFMR facility at EMSL for useful discussions. We also thank K. Baker for protein purification and characterization, X. Jia for assistance with the Varian 500 MHz spectrometer and P. Crescenti for manuscript preparation. This work was funded by the National Institutes of Health NCI, NIHLB, USAMRDC Breast Cancer Program, the University of California Breast Cancer Research Program, the State of California Cancer Research Program, the Susan G. Komen Breast Cancer Foundation, the Human Frontier Science Program and CaPCURE.
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Briknarová, K., Takayama, S., Brive, L. et al. Structural analysis of BAG1 cochaperone and its interactions with Hsc70 heat shock protein. Nat Struct Mol Biol 8, 349–352 (2001). https://doi.org/10.1038/86236
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DOI: https://doi.org/10.1038/86236
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