Laura Moullintraffort1, Matthieu Bruneaux2, Alexis Nazabal3, Diane Allegro, Emmanuel Giudice1, Franck Zal2, Vincent Peyrot4, Pascale Barbier4, Daniel Thomas1, and Cyrille Garnier1
- Structure et Dynamique des Macromolecules, UMR-CNRS 6026, Université de Rennes 1, 35042 Rennes Cedex France
- The Equipe Ecophysiologie des Invertébrés Marins des Milieux Extreˆmes, Université Pierre et Marie Curie Paris VI, CNRS UMR 7144, Station Biologique de Roscoff, B.P. 74, 29682 Roscoff, France
- CovalX AG, 8952 Zürich-Schlieren, Switzerland
- CRO2 UMR Inserm 911, Université de la Méditerranée, Faculté de Pharmacie, 13385 Marseille Cedex 5, France
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 Mg2+-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.
CovalX Technology Used
STI (1.21 mg/ml), STII (0.85 mg/ml), and Type III SEC-HPLC individually eluted fractions 129 (0.665 mg/ml), 130 (1.16 mg/ml), and 131 (2.42 mg/ml) were diluted in the CovalX K200 Stabilization Kit from 1/2 to 1/64 in a 10 μL of final volume. Control experiments were created in a 1:1 ratio with 1 μl of each mixture mixed with 1 μl of matrix (sinapic acid (10 mg/ml) in acetonitrile/water (1:1, v/v), TFA 0.1%). 1 μl of the mixture was sampled and spotted on a MALDI plate before being allowed to crystallize at room temperature. The remaining 9 μl of each sample were cross-linked by mixing in 1 μl of the CovalX K200 (2 mg/ml) before being incubated at room temperature for a variety of times (1,3 or 6 hours). The samples were analyzed by a mass spectrometer that had been modified with a CovalX HM2 detection system.