Binding of Human Nucleotide Exchange Factors to Heat Shock Protein 70 (Hsp70) Generates Functionally Distinct Complexes In Vitro [Protein Structure and Folding]
December 6th, 2013 by Rauch, J. N., Gestwicki, J. E.
Proteins with Bcl2-associated anthanogene (BAG) domains act as nucleotide exchange factors (NEFs) for the molecular chaperone, heat shock protein 70 (Hsp70). There are six BAG-family NEFs in humans and each is thought to link Hsp70 to a distinct cellular pathway. However, little is known about how the NEFs compete for binding to Hsp70 or how they might differentially shape its biochemical activities. Towards these questions, we measured binding of human Hsp72 (HSPA1A) to BAG1, BAG2, BAG3 and the unrelated NEF, Hsp105. These studies revealed a clear hierarchy of affinities: BAG3 > BAG1 > Hsp105 >> BAG2. All of the NEFs competed for binding to Hsp70 and their relative affinity values predicted their potency in nucleotide and peptide release assays. Finally, we combined the Hsp70-NEF pairs with co-chaperones of the J protein family, DnaJA1, DnaJA2, DnaJB1 and DnaJB4, to generate sixteen permutations. The activity of the combinations in ATPase and luciferase refolding assays were dependent on the identity and stoichiometry of both the J protein and NEF, such that some combinations were potent chaperones, whereas others were inactive. Given the number and diversity of co-chaperones in mammals, it is likely that combinatorial assembly could generate a large number of distinct permutations.