Client Proteins and Small Molecule Inhibitors Display Distinct Binding Preferences for Constitutive and Stress-Induced HSP90 Isoforms and Their Conformationally Restricted Mutants

Abstract

The two cytosolic/nuclear isoforms of the molecular chaperone HSP90, stress-inducible HSP90α and constitutively expressed HSP90β, fold, assemble and maintain the three-dimensional structure of numerous client proteins. Because many HSP90 clients are important in cancer, several HSP90 inhibitors have been evaluated in the clinic. However, little is known concerning possible unique isoform or conformational preferences of either individual HSP90 clients or inhibitors. In this report, we compare the relative interaction strength of both HSP90α and HSP90β with the transcription factors HSF1 and HIF1α, the kinases ERBB2 and MET, the E3-ubiquitin ligases KEAP1 and RHOBTB2, and the HSP90 inhibitors geldanamycin and ganetespib. We observed unexpected differences in relative client and drug preferences for the two HSP90 isoforms, with HSP90α binding each client protein with greater apparent affinity compared to HSP90β, while HSP90β bound each inhibitor with greater relative interaction strength compared to HSP90α. Stable HSP90 interaction was associated with reduced client activity. Using a defined set of HSP90 conformational mutants, we found that some clients interact strongly with a single, ATP-stabilized HSP90 conformation, only transiently populated during the dynamic HSP90 chaperone cycle, while other clients interact equally with multiple HSP90 conformations. These data suggest different functional requirements among HSP90 clientele that, for some clients, are likely to be ATP-independent. Lastly, the two inhibitors examined, although sharing the same binding site, were differentially able to access distinct HSP90 conformational states.

Gambogic acid identifies an isoform-specific druggable pocket in the middle domain of Hsp90β

“…To determine the relative binding strength of Bio-GBA to Hsp90 isoforms and their mutants, we followed a modified version of the LUMIER method, as previously described (8). Briefly, HEK293 cells were transfected with each 3×F-Hsp90 expression plasmid.…”

“…There are two major cytoplasmic isoforms of Hsp90: the stress-inducible Hsp90α and the constitutively expressed Hsp90β. These isoforms share 85% sequence identity and display significant functional redundancy, although each paralog has also developed distinct cellular functions during evolution (7,8). Unfortunately, current pharmacologic approaches do not allow interrogation of isoformspecific function, because currently available agents are not able to clearly distinguish between Hsp90α and Hsp90β (9).…”

Chemical Tools to Investigate Mechanisms Associated with HSP90 and HSP70 in Disease

“…For example, we know that solid support immobilized versions of several such inhibitors precipitate distinct HSP90 pools (Moulick et al, 2011, Prince et al, 2015). 17-AAG and NVP-AUY-922 but not PU-H71 (Figure 1A) augment the phenotype observed when cancer cells are treated with a compound that disrupts the HSP90-HOP interaction (Pimienta et al, 2011).…”

“…Lastly, using a defined set of HSP90 conformational mutants, the authors found that some clients interact strongly with a single, ATP-stabilized HSP90 conformation, only transiently populated during the dynamic HSP90 chaperone cycle, while other clients interact equally with multiple HSP90 conformations. GM and STA9090 (ganetespib) were differentially able to access these HSP90 conformational states (Prince et al, 2015). Taldone et al used an FITC-labeled HSP90 probe coupled with fluorescence polarization and combined it with computational analyses to rationalize such differences at the structural level (Taldone et al, 2013a).…”

Targeting the Diabetic Chaperome to Improve Peripheral Neuropathy

“…N-terminal Hsp90 inhibitors can also show a preferential interaction with SUMOylated versus non-SUMOylated Hsp90, resulting in greater activation of a HSR [42•]. Lastly, drug efficacy may also be influenced by the isoform ratio of chaperone complexes since N-terminal inhibitors can bind Hsp90β with greater affinity than Hsp90α [43]. Collectively, these data raise the possibility that the onset of disease may increase or possibly negate the therapeutic potential of various inhibitors by altering their recognition or interaction with Hsp90 isoforms and their heteroprotein complexes.…”