Biochemical and Biophysical Characterization of the Mg2+-induced 90-kDa Heat Shock Protein Oligomers

Abstract

The 90-kDa heat shock protein (Hsp90) is involved in the regulation and activation of numerous client proteins essential for diverse functions such as cell growth and differentiation. Although the function of cytosolic Hsp90 is dependent on a battery of cochaperone proteins regulating both its ATPase activity and its interaction with client proteins, little is known about the real Hsp90 molecular mechanism. Besides its highly flexible dimeric state, Hsp90 is able to self-oligomerize in the presence of divalent cations or under heat shock. In addition to dimers, oligomers exhibit a chaperone activity. In this work, we focused on Mg 2؉ -induced oligomers that we named Type I, Type II, and Type III in increasing molecular mass order. After stabilization of these quaternary structures, we optimized a purification protocol. Combining analytical ultracentrifugation, size exclusion chromatography coupled to multiangle laser light scattering, and high mass matrix-assisted laser desorption/ionization time of flight mass spectrometry, we determined biochemical and biophysical characteristics of each Hsp90 oligomer. We demonstrate that Type I oligomer is a tetramer, and Type II is an hexamer, whereas Type III is a dodecamer. These even-numbered structures demonstrate that the building brick for oligomerization is the dimer up to the Type II, whereas Type III probably results from the association of two Type II. Moreover, the Type II oligomer structure, studied by negative stain transmission electron microscopy tomography, exhibits a "nest-like" shape that forms a "cozy chaperoning chamber" where the client protein folding/protection could occur.Molecular chaperones are essential for the correct folding of neo-synthesized proteins in vivo, from their emergence from the ribosomal tunnel to their acquisition of a functional state. Among chaperone proteins, the 90-kDa heat shock protein (Hsp90) 2 is one of the most abundant proteins in the cytosol and is essential for cell survival (1). The Hsp90 functions require the cooperation of cochaperones and other chaperones within macro complexes (2). In contrast to most of the chaperone protein families, Hsp90 is not involved in de novo folding but operates at later stages of the folding process, assisting the maturation and activation of numerous client proteins, including steroid receptors and cell cycle kinases (3, 4). The effectiveness of Hsp90 in protecting and activating such proteins has led researchers to focus on the Hsp90 hub as a target for anticancer drugs (5-7). Hsp90 is ubiquitous and shows an extraordinary conservation from bacteria to higher eukaryotes. In the cell, Hsp90 amounts to 1-2% of the total proteins under nonstress conditions depending on the organism and cell type (2-25 mg/ml). Hsp90 is overexpressed under stress conditions such as heat or hypoxia (8 -10). In eukaryotes, the cytosol contains two isoforms, ␣ and ␤, encoded by distinct genes (11). These isoforms coexist in the cytosol; Hsp90␤ is constitutively expressed, whereas Hsp90␣ is stress-inducible. Th...

Hsp90 oligomerization process: How can p23 drive the chaperone machineries?

“…Hsp90 is able to self-associate both in vivo [42] and in vitro in the presence of divalent-cations [42,43,47]. We previously demonstrated that the Hsp90 oligomerization process involves the dimer as the building unit to form the tetramer and hexamer and that this latter, that we named the "cozy nest", is the building block for dodecamers.…”

“…As we wanted to isolate functional Hsp90:p23 complexes, all samples were prepared in the presence of the nonhydrolysable AMP-PNP nucleotide. Moreover, as Hsp90 complexes are highly dynamic, we chemically stabilized Hsp90:p23 complexes for purification purposes using our previously optimized cross-linking protocol with the zero-length EDC cross-linker [47]. The elution profile of the cross-linked p23 alone was clearly bimodal, suggesting the stabilization of p23 dimeric species.…”

“…Since the 62.0-64.5 mL pool corresponding to the [8,47] and the measured mass of 21.1 kDa for p23 (Fig. 6).…”

“…The elution profile of p23 [47]. d s 20,w was determined using linear regressions [43,47] at concentrations corresponding to major species found in L, H, and SH pools, respectively. ⁎⁎ Data not shown.…”

“…This latter would present an ideal and suitable environment for client protein accommodation and folding. Dodecamers then result from the association of two hexamers [47]. In spite of recent works, the involvement of Hsp90's domains in oligomerization process remains unclear as MC-HSP90 exists in three major states (dimer, tetramer and hexamer), and as NTDs could also participate in this process as they are able to self associate too [48].…”