BAG-1, a negative regulator of Hsp70 chaperone activity, uncouples nucleotide hydrolysis from substrate release

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Abstract
Molecular chaperones influence the process of protein folding and, under conditions of stress, recognize non‐native proteins to ensure that misfolded proteins neither appear nor accumulate. BAG‐1, identified as an Hsp70 associated protein, was shown to have the unique properties of a negative regulator of Hsp70. Here, we demonstrate that BAG‐1 inhibits the in vitro protein refolding activity of Hsp70 by forming stable ternary complexes with non‐native substrates that do not release even in the presence of nucleotide and the co‐chaperone, Hdj‐1. However, the substrate in the BAG‐1‐containing ternary complex does not aggregate and remains in a soluble intermediate folded state, indistinguishable from the refolding‐competent substrate–Hsp70 complex. BAG‐1 neither inhibits the Hsp70 ATPase, nor has the properties of a nucleotide exchange factor; instead, it stimulates ATPase activity, similar to that observed for Hdj‐1, but with opposite consequences. In the presence of BAG‐1, the conformation of Hsp70 is altered such that the substrate binding domain becomes less accessible to protease digestion, even in the presence of nucleotide and Hdj‐1. These results suggest a mechanistic basis for BAG‐1 as a negative regulator of the Hsp70–Hdj‐1 chaperone cycle.