Physicochemical Properties of the Mammalian Molecular Chaperone HSP60

Abstract

The E. coli GroEL/GroES chaperonin complex acts as a folding cage by producing a bullet-like asymmetric complex, and GroEL exists as double rings regardless of the presence of adenosine triphosphate (ATP). Its mammalian chaperonin homolog, heat shock protein, HSP60, and co-chaperonin, HSP10, play an essential role in protein folding by capturing unfolded proteins in the HSP60/HSP10 complex. However, the structural transition in ATPase-dependent reaction cycle has remained unclear. We found nucleotide-dependent association and dissociation of the HSP60/HSP10 complex using various analytical techniques under near physiological conditions. Our results showed that HSP60 exist as a significant number of double-ring complexes (football- and bullet-type complexes) and a small number of single-ring complexes in the presence of ATP and HSP10. HSP10 binds to HSP60 in the presence of ATP, which increased the HSP60 double-ring formation. After ATP is hydrolyzed to Adenosine diphosphate (ADP), HSP60 released the HSP10 and the dissociation of the double-ring to single-rings occurred. These results indicated that HSP60/HSP10 undergoes an ATP-dependent transition between the single- and double-rings in their system that is highly distinctive from the GroEL/GroES system particularly in the manner of complex formation and the roles of ATP binding and hydrolysis in the reaction cycle.

Keywords: GroEL; HSP60; chaperonin; molecular chaperone.

Figure 1

Transmission electron microscopy observation of chaperonin complex. (A) Transmission electron microscopic analysis of heat shock protein, HSP60 (upper) or HSP60/HSP10 (lower) in the presence (right) or absence (left) of 1 mM ATP. White arrows, white arrowheads and black arrowheads indicate side views of single rings (HSP60₇), double rings (HSP60₁₄) and football-type complexes (HSP60₁₄-HSP10₁₄), respectively; (B) Classification of five shapes of HSP60/HSP10 complex observed in the presence of 1 mM adenosine triphosphate (ATP). More than 500 side views of HSP60 complexes were counted for each experiment. Mean and standard deviations of three independent experiments are shown; (C) HSP60 alone (left) or HSP60/HSP10 (right) in the presence of 1 mM adenosine diphosphate (ADP). White arrows indicate side views of single rings (HSP60₇). (HSP10/single ring HSP60 ratio = 2); (D) GroEL with GroES in the presence (right) and absence (left) of 1 mM ATP. Black arrows and arrowheads indicate double rings (GroEL₁₄) and bullet-type (GroEL₁₄-GroES₇), respectively. (GroES/double ring GroEL ratio = 4).

Figure 2

Small angle X-ray scattering (SAXS) analysis of HSP60/HSP10 and GroEL/GroES complex under the various nucleotide conditions. (A,B) SAXS patterns of HSP60 and GroEL under various conditions. Scattering intensities of HSP60 (A) and GroEL (B) with or without their co-chaperones in the presence and absence of 1mM ATP. Co-chaperone (single ring)/chaperonin (as double ring) ratio = 2; (C) SAXS patterns of HSP60/HSP10 in the presence of various nucleotides including ATP, ADP, Adenosine-5′-(β, γ-imido)-triphosphate (AMP-PNP) and Adenosine 5′-(γ-thio)-triphosphate (ATPγS); (D) I(0)/C values of HSP60 (solid circle) and GroEL (open circle) in the presence of various concentrations of co-chaperones under the nucleotide-free (Blue), 1 mM ATP (Red) or ADP (Green) conditions; (E) R_g values of HSP60 (solid circle) and GroEL (open circle) in the presence of various concentrations of co-chaperones under the conditions indicated were analyzed by a Guinier plot. Protein concentrations were 3 mg/mL HSP60 and 0.15–2.25 mg/mL HSP10, or 3 mg/mL GroEL and 0.15–2.25 mg/mL GroES when cochaperone/chaperonin (single ring) ratio = 1:1; (F) I(0)/C D_max values of HSP60 (solid circle) and GroEL (open circle) in the presence of various concentrations of co-chaperones under the nucleotide-free (Blue), 1 mM ATP (Red) or ADP (Green) conditions.

Figure 3

Association of HSP10 to HSP60 under various nucleotide conditions. (A) Trypsin resistance of HSP60/HSP10 or GroEL/GroES under nucleotide-free, 1 mM ATP, ADP, AMP-PNP or ATPγS condition. Digested samples were analyzed by SDS-PAGE (12% gels). White and black arrowheads indicate HSP60/GroEL and HSP10/GroES positions, respectively; (B) FCCS analysis of the association between HSP60 and HSP10. Cross-correlation functions of Atto 647N-labeled HSP60 and Atto 488-labeled HSP10 under nucleotide-free (dark grey), 1 mM ATP (red), ADP (blue), AMP-PNP (yellow), ATPγS (green) condition, or both Atto 488- and Atto 647N-labeled HSP60 as a positive control (light grey) are indicated.

Figure 4

The formation of stable football complex of HSP60(D398A)/HSP10/ATP. (A) ATPase activity of wild type HSP60, HSP60/HSP10 or D398A mutant of HSP60 (0.1 μM). (B) Protease sensitivity assay of HSP10 without HSP60, with wild-type HSP60 or HSP60(D398A) in the presence of 1 mM ATP. The values were quantified from stained bands of HSP10 in SDS-PAGE gel. (C) Native-PAGE analysis of wild type HSP60, HSP60(D398A), or GroEL with or without their co-chaperone in the presence of ATP. Native-PAGE gel and buffer were supplemented with 1 mM ATP. The arrow indicates the putative complex of HSP60(D398A)/HSP10/ATP. (D) Electron microscopic analysis of HSP60(D398A)/HSP10 in the presence of ATP. White arrows indicate side views of HSP60(D398A)/HSP10 football-type complexes.

Figure 4

The formation of stable football complex of HSP60(D398A)/HSP10/ATP. (A) ATPase activity of wild type HSP60, HSP60/HSP10 or D398A mutant of HSP60 (0.1 μM). (B) Protease sensitivity assay of HSP10 without HSP60, with wild-type HSP60 or HSP60(D398A) in the presence of 1 mM ATP. The values were quantified from stained bands of HSP10 in SDS-PAGE gel. (C) Native-PAGE analysis of wild type HSP60, HSP60(D398A), or GroEL with or without their co-chaperone in the presence of ATP. Native-PAGE gel and buffer were supplemented with 1 mM ATP. The arrow indicates the putative complex of HSP60(D398A)/HSP10/ATP. (D) Electron microscopic analysis of HSP60(D398A)/HSP10 in the presence of ATP. White arrows indicate side views of HSP60(D398A)/HSP10 football-type complexes.

Figure 5

Rhodanese refolding assay to measure the folding cavity formation of chaperonin. (A) Rhodanese activity by chaperonins after 60 min (n = 3). HSP60/HSP10, HSP60(D398A)/HSP10, and GroEL/GroES are used. Hexokinase was added at 3 s after the initiation of the folding reaction. The time course of rhodanere refolding was shown in Figure S6. (B) Electron microscopic analysis of HSP60/HSP10 complex after hexokinase treatment. White arrows indicate side views of HSP60 single-ring.

Figure 6

The proposed reaction scheme of human HSP60/HSP10. (a) Single-ring state formed by dissociation of HSP10 and ADP after ATP hydrolysis in the bullet-type complex in (d). (b) The binding of ATP and HSP10 to HSP60 to form single-ring HSP60/HSP10/ATP complex. (c) The formation of the double-ring football complex by association of two HSP60/HSP10/ATP single-rings. (d) The formation of the bullet-type complex by dissociation of HSP10 and ADP after ATP hydrolysis in one of the rings.