On the mechanism of chaperone activity of the small heat-shock protein of Methanococcus jannaschii

Abstract

The small heat-shock protein (sHSP) from Methanococcus jannaschii (Mj HSP16.5) forms a homomeric complex of 24 subunits and has an overall structure of a multiwindowed hollow sphere with an external diameter of approximately 120 A and an internal diameter of approximately 65 A with six square "windows" of approximately 17 A across and eight triangular windows of approximately 30 A across. This sHSP has been known to protect other proteins from thermal denaturation. Using purified single-chain monellin as a substrate and a series of methods such as protease digestion, antibody binding, and electron microscopy, we show that the substrates bind to Mj HSP16.5 at a high temperature (80 degrees C) on the outside surface of the sphere and are prevented from forming insoluble substrate aggregates in vitro. Circular dichroism studies suggest that a very small, if any, conformational change occurs in sHSP even at 80 degrees C, but substantial conformational changes of the substrate are required for complex formation at 80 degrees C. Furthermore, deletion mutation studies of Mj HSP16.5 suggest that the N-terminal region of the protein has no structural role but may play an important kinetic role in the assembly of the sphere by "preassembly condensation" of multiple monomers before final assembly of the sphere.

Fig. 1.

SEC was performed by using a TosoHaas TSK G4000SW column as described in Materials and Methods. Squares, Mj HSP16.5–SCM at a 1:1 molar ratio, unheated; diamonds, Mj HSP16.5–SCM at a 1:1 molar ratio was heated at 80°C for 20 min and centrifuged at 10,000 × g for 10 min, and the supernatant was applied to the sizing column.

Fig. 2.

Electron micrographs and averaged particle images of Mj HSP16.5 and Mj HSP16.5 complexed with SCM, a substrate protein, prepared as described in Materials and Methods. (A) Mj HSP16.5. (B) Mj HSP16.5 in complex with SCM. (Scale bar, 100 nm.) (C and D) Gallery of single-particle images selected from a computer graphics display of the micrographs of A and B, respectively.

Fig. 3.

Proteinase K susceptibility. Lane 1, molecular mass standards; lane 2, SCM–Mj HSP16.5 at a 1:1 ratio, unheated; lane 3, SCM–Mj HSP16.5 at a 1:1 ratio, heated at 80°C for 20 min and centrifuged (supernatant was incubated with proteinase K as described in Materials and Methods); lane 4, Mj HSP16.5 was heated at 80°C for 20 min and then treated with proteinase K; lane 5, SCM was heated at 60°C for 20 min and then treated with proteinase K.

Fig. 4.

Western blot of SCM–Mj HSP16.5 complex. (A) A 0.8% native agarose gel was run as described in Materials and Methods. Lane 1, SCM, 5 μg; lane 2, Mj HSP16.5, 7.4 μg, was heated at 80°C for 20 min and centrifuged for 5 min at 14,000 × g, and the supernatant was loaded; lane 3, SCM–Mj HSP16.5 (1:1 molar ratio) was heated at 80°C for 20 min and centrifuged for 5 min at 14,000 × g, and the supernatant was loaded. (B) A portion of the gel from A was transferred onto nitrocellulose paper and probed with polyclonal antiserum raised against SCM. Lane 1, SCM, 5 μg; lane 3, Mj HSP16.5, 7.4 μg, was heated at 80°C for 20 min and centrifuged for 5 min at 14,000 × g, and the supernatant was loaded; lane 5, SCM–Mj HSP16.5 (1:1 molar ratio) was heated at 80°C for 20 min and centrifuged for 5 min at 14,000 × g, and the supernatant was loaded.

Fig. 5.

CD spectra for SCM and Mj HSP16.5. (A) CD spectra of SCM at 25°C. (B) Thermal melting of SCM. The CD signal at 218 nm is plotted as a function of temperature. (C) CD spectra of Mj HSP16.5 at 25°C. (D) Thermal melting of Mj HSP16.5. The CD signal at 218 nm is plotted as a solid line; at 220 nm it is plotted as a dashed line.

Fig. 6.

CD spectra before and after mixing SCM and Mj HSP16.5. (A) CD spectra of SCM and Mj HSP16.5 at a 1:1 molar ratio at 25°C. Dashed line, before mixing, where two solutions are separated by a divider; solid line, after mixing and heating. (B) Thermal melting of SCM–Mj HSP16.5 at a 1:1 molar ratio. Dashed line, before mixing; solid line, after mixing. The CD signal at 218 nm is plotted as a function of temperature.

Fig. 7.

Model for assembly of Mj HSP16.5. The N-terminal region of Mj HSP16.5 acts as a hydrophobic tether of the folded α-crystalline domain. Many such tethered α-crystalline domains are brought together by the hydrophobic “glue” of the tethers to form a large multimer, a preassembly condensation process. With many subunits in proximity, the final assembly of the sphere is facilitated.

Fig. 8.

Schematic representation of the interaction of protein substrate with Mj HSP16.5. In the absence of sHSP, stress induces proteins to partially denature and expose hydrophobic regions that stick together to form protein aggregates and precipitation. In the presence of sHSP, these partially denatured proteins are attracted to the surface of the sphere and are prevented from aggregating to themselves. After release of stress, the substrate is released from the sphere and refolded.