Modulation of Chaperone Function and Cochaperone Interaction by Novobiocin in the C-terminal Domain of Hsp90

Abstract

The C-terminal domain of Hsp90 displays independent chaperone activity, mediates dimerization, and contains the MEEVD motif essential for interaction with tetratricopeptide repeat-containing immunophilin cochaperones assembled in mature steroid receptor complexes. An ␣-helical region, upstream of the MEEVD peptide, helps form the dimerization interface and includes a hydrophobic microdomain that contributes to the Hsp90 interaction with the immunophilin cochaperones and corresponds to the binding site for novobiocin, a coumarin-related Hsp90 inhibitor. Mutation of selected residues within the hydrophobic microdomain significantly impacted the chaperone function of a recombinant C-terminal Hsp90 fragment and novobiocin inhibited wild-type chaperone activity. Prior incubation of the Hsp90 fragment with novobiocin led to a direct blockade of immunophilin cochaperone binding. However, the drug had little influence on the pre-formed Hsp90-immunophilin complex, suggesting that bound cochaperones mask the novobiocin-binding site. We observed a differential effect of the drug on Hsp90-immunophilin interaction, suggesting that the immunophilins make distinct contacts within the C-terminal domain to specifically modulate Hsp90 function. Novobiocin also precluded the interaction of full-length Hsp90 with the p50 There is now increasing evidence that receptor function is critically dependent on the selection of immunophilin within steroid receptor complexes (2-4). This may be governed, in part, by the selective preference of receptors for specific immunophilins. PP5 has been reported to have a modulating role in glucocorticoid signaling (5), and there are strong indications that FKBP51 inhibits glucocorticoid receptor function. Elevated expression of FKBP51, resulting in greatly increased incorporation of FKBP51 into glucocorticoid receptor complexes, reduces hormone-binding affinity and promotes glucocorticoid resistance in primates (6, 7). FKBP51 also sequesters glucocorticoid receptor within the cytoplasm (8, 9), but hormone binding induces a functional exchange of FKBP52 for FKBP51 in receptor complexes allowing translocation of the complex to the nucleus (8). In a yeast model, FKBP52 was shown to dramatically potentiate glucocorticoid-dependent reporter gene activity through a mechanism that results in increased receptor hormone-binding affinity (10). Consistent with previous findings, coexpression of FKBP51 blocked the potentiating effects of FKBP52. These potentiating properties require FKBP52 catalytic activity as well as a functional interaction of the immunophilin with Hsp90. Receptor function, then, can be directly influenced by the prolyl isomerase activity of a TPR immunophilin. The Smith laboratory has now extended the study of FKBP52 function to a FKBP52 knock-out mouse model (11-13). Male mice exhibit many features in common with partial androgen insensitivity, reflecting loss of FKBP52-mediated potentiation of androgen receptor function (11). Female mice display a maternal defect linked to progesterone ins...

Hsp90 Functions in the Targeting and Outer Membrane Translocation Steps of Tom70-mediated Mitochondrial Import

“…For a mechanistic comparison with NB, the effect of GA on import was also studied. Both drugs have been demonstrated to impair Hsp90 function, although much lower concentrations of GA are typically required: 0.5-10 M GA in live cells or 10 -100 M GA in vitro (48,(53)(54)(55)(56)(57)(58)(59)(60)(61). Such concentrations agree with the ϳ2 M affinity of GA for Hsp90 measured in vitro (53).…”

“…NB was first reported not to disrupt interactions between Hsp90 and the TPR domain co-chaperones FKBP52 (FK506-binding protein of 52 kDa) and protein phosphatase-5 (15). However, a recent report suggested that, under certain conditions, differential displacement of FKBP52 is observed (61). To investigate whether this is the case for Tom70, the direct interaction between Tom70 and Hsp90 was examined.…”

“…Also, it has been proposed that coumarin-derived antibiotics such as NB could affect the C-terminal dimerization of Hsp90 (60,61). To investigate these possibilities, the gel filtration behavior of Hsp90 itself was analyzed after different treatments.…”

Heat shock protein 90 (HSP90) contributes to cytosolic translocation of extracellular antigen for cross-presentation by dendritic cells

“…NB targets the C-terminal portion of the protein (23), which is critical for the formation of HSP90 dimers to exert its chaperone activity (24), whereas Rad and GA bind the N-terminal ATPbinding pocket to block the ATP-dependent chaperone cycling (25). On the basis of this information, we generated conditional HSP90α-null mice by floxing exons 9 and 10, which encode the C-terminal region of the protein (Fig.…”

Conformational dynamics of the molecular chaperone Hsp90

“…Novobiocin is a small molecule inhibitor that binds to the CTD of Hsp90 (Marcu et al, 2000a;Marcu et al, 2000b;Soti et al, 2002). Like GA and radicicol, novobiocin inhibits the formation of Hsp90 complexes with client or p23, but unlike GA it also reduces the affinity of Hsp90 for TPR-containing co-chaperones that bind to the CTD (Allan et al, 2006;Yun et al, 2004). The disruption of these Hsp90 complexes suggests that novobiocin causes structural rearrangements within Hsp90 that are distinct from the changes seen with GA. Epigallocatechin gallate (ECGG) the active ingredient found in green tea also directly binds to Hsp90 and acts as an AhR antagonist.…”