A proteomic investigation of ligand-dependent HSP90 complexes reveals CHORDC1 as a novel ADP-dependent HSP90-interacting protein

Abstract

Structural studies of the chaperone HSP90 have revealed that nucleotide and drug ligands induce several distinct conformational states; however, little is known how these conformations affect interactions with co-chaperones and client proteins. Here we use tandem affinity purification and LC-MS/MS to investigate the proteome-wide effects of ATP, ADP, and geldanamycin on the constituents of the human HSP90 interactome. We identified 52 known and novel components of HSP90 complexes that are regulated by these ligands, including several co-chaperones. Interestingly, our results also show that geldanamycin treatment causes HSP90 complexes to become significantly enriched for core transcription machinery, suggesting that HSP90 inhibition may have broad based effects on transcription and RNA processing. We further characterized a novel ADP-dependent HSP90 interaction with the cysteine- and histidine-rich domain (CHORD)-containing protein CHORDC1. We show that this interaction is stimulated by high ADP:ATP ratios in cell lysates and in vitro with purified recombinant proteins. Furthermore, we demonstrate that this interaction is dependent upon the ability of HSP90 to bind nucleotides and requires the presence of a linker region between the CHORD domains in CHORDC1. Together these findings suggest that the HSP90 interactome is dynamic with respect to nucleotide and drug ligands and that pharmacological inhibition of HSP90 may stimulate the formation of specific complexes.

Fig. 1.

Spectral abundance factors for HSP90-interacting proteins. a, N-terminal TAP-tagged HSP90 interactors. Data are averages of three replicate experiments. Experimental groups are color-coded: WT ATP (red), WT ADP (blue), E47A ATP (green), and WT GA (yellow). Proteins are arranged in descending order according to their spectral abundance factor. The horizontal axis displays the SAF, and the scales change for every block of proteins to account for the wide range of SAF values for this data set. Protein names are shown to the left. Statistical information including standard deviation is available in supplemental Table S1. Asterisks denote proteins that are not regulated by ligands (ANOVA p > 0.01) b, C-terminal TAP-tagged HSP90 interactors.

Fig. 2.

Mutations affecting nucleotide binding and TPR domain interactions diminish the effects of geldanamycin on HSP90 complexes. SAF values for all proteins indentified in purifications containing geldanamycin using N-terminal TAP-tagged HSP90 WT (yellow) and/or D93N (gray) and ΔMEEVD mutants (black) are shown. Proteins that did not pass the filtering criteria in either of the two mutant experimental groups are not shown. All data are averages from three replicate experiments. All statistical data including raw data and standard deviations are available in supplemental Table S1.

Fig. 3.

HSP90-CHORDC1 interaction is stimulated by high ADP:ATP ratios and is dependent upon elements within HSP90 and CHORDC1. a, Western blot (WB) of affinity-precipitated C-terminally TAP-tagged HSP90-CHORDC1 complexes in the presence of increasing amounts of ADP and decreasing ratios of ADP:ATP. Complexes were precipitated with streptavidin beads, washed, and subjected to SDS-PAGE. Blots were probed with streptavidin-horseradish peroxidase (HRP) (TAP-HSP90) and anti-HA antibodies (HA-CHORDC1). Vector only (VO) controls are shown in the lanes to the left, and loading controls are shown in the two panels below. Densitometry was used to quantify the CHORDC1 signal for the indicated ATP:ADP ratios. b, a schematic diagram of HSP90 and CHORDC1 truncation and point mutants used in this analysis. Numbers indicate the amino acid residues. c, Western blot of HA-tagged full-length CHORDC1 affinity-precipitated with the indicated C-terminally TAP-tagged HSP90α mutants in the presence of 1 mm ATP or 1 mm ADP. Complexes were purified and probed as in a. Vector only controls are shown in the two lanes to the extreme right. d, Western blot of HA-tagged CHORDC1 truncation mutants affinity-precipitated with TAP-tagged full-length wild type HSP90α in the presence of 1 mm ATP and 1 mm ADP and probed as in a. Vector only controls are shown in the two lanes to the extreme right. AP, affinity precipitation.

Fig. 4.

CHORDC1 interacts with HSP90 in vitro in an ADP-dependent manner. 1 μg of purified HSP90-GST WT, D93N, and E47A fusions immobilized on glutathione beads was incubated with 100 ng of HA-CHORDC1 for 5 h at 4 °C in the presence of 5 mm ATP or ADP or no nucleotides. Complexes were washed, subjected to SDS-PAGE, and immunoblotted with either anti-HA, anti-GST, or anti-HSP90 antibodies. The double band for HSP90 in the anti-GST and anti-HSP90 immunoblots represents a breakdown product of HSP90. AP, affinity precipitation; WB, Western blot.