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A chemical cross-linking method for the analysis of binding partners of heat shock protein-90 in intact cells
Shaoming Song1, Sutapa Kole2, and Michel Bernier1
1Laboratory of Clinical Investigation, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA
2Kelly Government Solutions, Rockville, MD, USA
BioTechniques, Vol. , No. , April 2012, pp. 1–7
Full Text (PDF)
Method Summary

Here, we demonstrate chemical cross-linking of the 240-kDa heteroconjugate of the Hsp90 molecular chaperone using ethylene glycolbis (succinimidylsuccinate) in intact cells. Use of the cross-linking agent will enable the further study of Hsp90 function and interactions.


Members of the heat shock protein-90 (Hsp90) family are key regulators of biological processes through dynamic interaction with a multitude of protein partners. However, the transient nature of these interactions hinders the identification of Hsp90 interactors. Here we show that chemical cross-linking with ethylene glycolbis (succinimidylsuccinate), but not shorter cross-linkers, generated an abundant 240-kDa heteroconjugate of the molecular chaperone Hsp90 in different cell types. The combined use of pharmacological and genetic approaches allowed the characterization of the subunit composition and subcellular compartmentalization of the multimeric protein complex, termed p240. The in situ formation of p240 did not require the N-terminal domain or the ATPase activity of Hsp90. Utilizing subcellular fractionation techniques and a cell-impermeant cross-linker, subpopulations of p240 were found to be present in both the plasma membrane and the mitochondria. The Hsp90-interacting proteins, including Hsp70, p60Hop and the scaffolding protein filamin A, had no role in governing the formation of p240. Therefore, chemical cross-linking combined with proteomic methods has the potential to unravel the protein components of this p240 complex and, more importantly, may provide an approach to expand the range of tools available to the study of the Hsp90 interactome.

Members of the 90-kDa heat shock protein (Hsp90) family are molecular chaperones with an intrinsic ATPase activity that functions in the folding, maturation, and activation of large number of protein interactors known as clients (for current list of client proteins, see: These client proteins have critical roles in a diverse range of biological processes, including signal transduction, cellular trafficking, metabolism, transcription, and cell growth and differentiation. Hsp90 exists in a homodimeric state in association with several co-chaperones and accessory proteins that regulate the ATPase activity of Hsp90 and ensure client protein assembly into multiprotein complexes (1,2). Structural studies and molecular modeling methods have provided a model in which the dynamic conformational changes between the nucleotide-free and the nucleotide-bound forms of Hsp90 enable spatiotemporal recruitment of select co-chaperones while effecting transport and/or assembly of functional signaling complexes (3-5). Hsp90 has three distinct domains, termed N-terminal domain, which contains an ATP binding site, the highly charged middle domain that has high affinity for co-chaperones and client proteins, and the C-terminal domain harboring a second ATP binding site and a conserved pentapeptide sequence recognized by co-chaperones. The N-terminal nucle-otide binding pocket has been shown to bind the ansamycin antibiotic geldanamycin, whereas the coumarin antibiotic novobiocin and some chemotherapeutic agents interact with the C-terminal ATP binding site to prevent hydrolysis of ATP. As a result, these pharmacological inhibitors disrupt the chaperone activity of Hsp90, causing ubiquitinylation and subsequent degradation of a subset of pro-oncogenic client proteins (6,7).

The study of Kang et al. (8) has shown that unlike most normal tissues, mitochondria of various tumors contain Hsp90, where it interacts with the immunophilin cyclophilin D to confer anti-apoptotic protection (9). In addition to being found intracellularly,Hsp90 is present at the cell membrane and participates in a multitude of extracellular functions, from acting in skin cell motility and wound healing (10), to cancer cell invasion (11,12). Extracellular Hsp90 binds the surface low density lipoprotein (LDL) receptor-related protein 1/CD91, matrix metalloproteinase 2, and the extracellular domain of HER-2 (11-13).

The human Hsp90 interactome has been characterized recently using immunopurification and affinity capture with immobilized Hsp90 (14-16). Several co-chaperones and client proteins were identified; however, these experiments were not designed to study protein interactions in intact cells, as many of these interactions with Hsp90 are relatively transient due to their dependence of the dynamic ATP-dependent cycle (17,18) and cellular redox state (19,20). Here, we use a method for chemical cross-linking to evaluate the formation of heteroconjugates of Hsp90 in intact cells. When compared with the mammalian two-hybrid system and classical proteomic approach, the protein cross-linking technique has the advantage to stabilize covalently Hsp90-interactor protein complexes, thereby minimizing the possibility of the native complexes undergoing dissociation during cell lysis and biochemical fractionation. This approach has enabled the detection of an abundant Hsp90 multimeric complex, termed p240, in various subcellular compartments, including mitochondria and the plasma membrane facing the extracellular environment.

Materials and methods


The following Hsp90 inhibitors, 17-allylamino-17-demethoxygeldanamycin (17-AAG), 17-desmethoxy-17-N,N-dimethylaminoethylamino-geldanamycin (17-DMAG), and novobiocin, were purchased from EMD Biosciences (Gibbstown, NJ, USA). The homobifunctional protein cross-linkers dimethyl pimelimidate (DMP), disuccinimidyl suberate (DSS), ethylene glycolbis(succinimidylsuccinate) (EGS), and the water soluble ethylene glycolbis(sulfosuccinimidylsuccinate) (Sulfo-EGS) were from Thermo Scientific Pierce (Rockford, IL, USA). Novex tris-glycine gels (4%–12% and 6%), iBlot apparatus, and Opti-MEM were from Invitrogen (Carlsbad, CA, USA). Non-fat dry milk and Tween-20 were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Tris-buffered saline was from ScyTek Laboratories (Logan, UT, USA). Mouse anti-human Hsp90α/β antibody was purchased from BD Biosciences (cat. 610419; San Jose, CA, USA); monoclonal antibodies against Hsp70 (sc32239) and GAPDH (sc32233), and polyclonal anti-IκBα (sc371) and anti-EGFR (sc03) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-HSF1 (clone 10H8) and p60Hop (clone DS14F5) monoclonal antibodies were from Enzo Life Sciences (Farmingdale, NY, USA); mitochondrial complex V antibody was from Molecular Probes, Invitrogen (cat. 439800; Eugene, OR, USA), while Stratagene anti-FLAG M2 clone and anti-filamin A (FLNa) were from Agilent Technology (cat. 200471; Cedar Creek, TX, USA) and Fitzgerald Industries International (cat. 10-F82A; Acton, MA, USA), respectively. Protein G agarose was purchased from Millipore (Temecula, CA, USA). Enhanced chemiluminescent (ECL) detection system, donkey anti-rabbit horseradish peroxidase-linked IgG, and sheep anti-mouse horseradish peroxidase-linked IgG were from GE Healthcare/Amersham Biosciences (Piscataway, NJ, USA). α-Minimal essential medium (α-MEM) was purchased from Invitrogen, and Ham's F12 medium was from Cellgro/Mediatech (Manassas, VA, USA). Human FLNa small interfering RNA (siRNA), Qiagen mini- and maxi-plasmid preparation kits, and Qiaquick PCR kit were from Qiagen (Valencia, CA, USA). Lipofectamine RNAiMAX reagent and Lipofectamine 2000 transfection reagent were purchased from Invitrogen.

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