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.
Highlights
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
Native-PAGE analysis of Hsp90 preincubated in the presence of magnesium showed a major band for the dimer and very low intensity bands corresponding to oligomers (Fig. 1A, lane 1)
When Hsp90 was cross-linked in the presence of magnesium, native-PAGE revealed a low intensity band for the Hsp90 dimer and at least three or more additional bands of higher molecular masses corresponding to magnesium-induced oligomers (Fig. 1A, lane 2)
Summary
Hsp was purified from pig brains according to the method of Yonezawa et al [31] modified by Garnier et al [23, 32]. Large Scale Purification by Size Exclusion Chromatography— A 500-l cross-linked and quenched sample (final concentration, 29 M) was loaded on a Superdex 200 size exclusion chromatography column, 1 cm ϫ 210 cm (90/60/30/30 cm Tricorn columns in series, GE Healthcare) equilibrated in 10 mM MESNaOH, 8 mM MgCl2, 150 mM NaCl, pH 6.5, with chromatography conditions controlled by an HPLC pump system (625 LC System, Waters). The samples were supplemented with Laemmli buffer [36] and loaded onto polyacrylamide 3– 8% gradient gel. The samples were supplemented with buffer containing Coomassie Blue G-250 in 30% glycerol and loaded onto 6/8% double layer polyacrylamide gels. Migrations were performed in Tris-glycine buffer over 2.5 or 3 h at 20 mA/gel.
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