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While cytosolic Hsp90 chaperones have been extensively studied, less is known about how the ER Hsp90 paralog Grp94 recognizes clients and influences client folding. Here, we examine how Grp94 and the ER Hsp70 paralog, BiP, influence the folding of insulin-like growth factor 2 (IGF2), an established client protein of Grp94. ProIGF2 is composed of a disulfide-bonded insulin-like hormone and a C-terminal E-peptide that has sequence characteristics of an intrinsically disordered region. BiP and Grp94 have a minimal influence on folding whereby both chaperones slow proIGF2 folding and do not substantially alter the disulfide-bonded folding intermediates, suggesting that BiP and Grp94 may have an additional influence unrelated to proIGF2 folding. Indeed, we made the unexpected discovery that the E-peptide region allows proIGF2 to form dynamic oligomers. ProIGF2 oligomers can transition from a dynamic state that is capable of exchanging monomers to an irreversibly aggregated state, providing a plausible role for BiP and Grp94 in regulating proIGF2 oligomerization. In contrast to the modest influence on folding, BiP and Grp94 have a stronger influence on proIGF2 oligomerization and these chaperones exert counteracting effects. BiP suppresses proIGF2 oligomerization while Grp94 can enhance proIGF2 oligomerization in a nucleotide-dependent manner. We propose that BiP and Grp94 regulate the assembly and dynamic behavior of proIGF2 oligomers, although the biological role of proIGF2 oligomerization is not yet known.

The ATPase cycle of the Hsp90 molecular chaperone is essential for maintaining the stability of numerous client proteins. Extensive analysis has focused on ATP-driven conformational changes of Hsp90; however, little is known about how Hsp90 operates under physiological nucleotide conditions in which both ATP and ADP are present. By quantifying Hsp90 activity under mixed nucleotide conditions, we find dramatic differences in ADP sensitivity among Hsp90 homologs. ADP acts as a strong ATPase inhibitor of cytosol-specific Hsp90 homologs, whereas organellular Hsp90 homologs (Grp94 and TRAP1) are relatively insensitive to the presence of ADP. These results imply that an ATP/ADP heterodimer of cytosolic Hsp90 is the predominant active state under physiological nucleotide conditions. ADP inhibition of human and yeast cytosolic Hsp90 can be relieved by the cochaperone aha1. ADP inhibition of bacterial Hsp90 can be relieved by bacterial Hsp70 and an activating client protein. These results suggest that altering ADP inhibition may be a mechanism of Hsp90 regulation. To determine the molecular origin of ADP inhibition, we identify residues that preferentially stabilize either ATP or ADP. Mutations at these sites can both increase and decrease ADP inhibition. An accounting of ADP is critically important for designing and interpreting experiments with Hsp90. For example, contaminating ADP is a confounding factor in fluorescence resonance energy transfer experiments measuring arm closure rates of Hsp90. Our observations suggest that ADP at physiological levels is important to Hsp90 structure, activity, and regulation.