HSP60 possesses a GTPase activity and mediates protein folding with HSP10

Abstract

The mammalian molecular chaperone, HSP60, plays an essential role in protein homeostasis through mediating protein folding and assembly. The structure and ATP-dependent function of HSP60 has been well established in recent studies. After ATP, GTP is the major cellular nucleotide. In this paper, we have investigated the role of GTP in the activity of HSP60. It was found that HSP60 has different properties with respect to allostery, complex formation and protein folding activity depending on the nucleoside triphosphate present. The presence of GTP slightly affected the ATPase activity of HSP60 during protein folding. These results provide clues as to the functional mechanism of the HSP60-HSP10 complex.

Figure 1

GTPase activity of HSP60 and physiological functions. (A), ATPase activity of HSP60 in the presence or absence of HSP10. The kinetic parameters were calculated by curve fitting to the Hill equation 1 or 2 in the Materials and Methods section by Kaleida Graph. (B), ATPase activity of HSP60 in the absence or presence of HSP10 in the low concentration range from 0 to 0.02 mM. The kinetic curves were directly fitted to the Hill equation 2. (C), GTPase activity of HSP60 in the presence or absence of HSP10 was calculated by curve fitting to the Hill equation 2. (D), Purified HSP60 was incubated with GTP at 37 °C for 2 h. Sample of time 0 and 2 h were separated by a C₁₈-reverse phase column and absorbance at 256 nm was recorded. (E), NTP-pull down assay was performed using ATP- or GTP-agarose in the presence of HSP60 or HSP10. In the current study, to stabilize the interaction between HSP60 and NTP-agarose, a pull down assay was performed in the presence of 0.2 mM AlCl₃ and 10 mM NaF. Samples were separated by 12% SDS-PAGE and detected by Coomassie Brilliant Blue R250-staining. (F), ATP- or GTP-agarose was eluted with 1 mM ATP or GTP in the presence of 0.2 mM AlCl₃ and 10 mM NaF, respectively.

Figure 2

Interaction between HSP60 and HSP10 induced by ATP- or GTPase activity. (A), The interaction between HSP60 and HSP10 in the presence of ATP or GTP was evaluated by a trypsin sensitivity assay. Samples were separated by 12% SDS-PAGE and protein bands were detected by Coomassie Brilliant Blue R250-staining. Asterisk indicates the proteolytic fragment of HSP10. (B), Refolding assay of the acid-denatured GFP was performed to investigate the interaction between HSP60 and HSP10. The fluorescence recovery of GFP at 535 nm with excitation at 485 nm was measured for 300 sec.

Figure 3

Oligomer-ization of HSP60 in the presence of ATP or GTP. (A), TEM images of HSP60 with or without HSP10 in the presence of ATP or GTP. Based on these images, statistical analysis was performed by counting 100 molecules (B). See also supplementary Figures S1A and S2C-E. C and D, SEC-MALS analysis of HSP60 with or without HSP10 in the presence of ATP or GTP. HSP60 with or without HSP10 was incubated for 5 min in the presence or absence of 1 mM nucleotides, then separated by gel filtration column chromatography. In the presence of nucleotides, the running buffer was treated with 1 mM ATP or GTP. The color of the detected Ri peak (solid line) and the molecular weight distribution (dashed line) corresponded. The molecular weight was calculated by Wyatt Astra Software.

Figure 4

Folding properties of HSP60/HSP10 complex in the presence of ATP or GTP. (A), Refolding assay of the chemical-denatured rhodanese. The refolding yield of rhodanese was calculated by measuring the recovery of the enzymatic activity. The refolding reaction was performed for 60 min and the rhodanese activity was measured at the indicated times. (B), Refolding assay of the chemical-denatured citrate synthase. The refolding reaction was performed for 60 min and the refolding yield was calculated by measuring the recovery of the enzymatic activity. The in-cage folding of the HSP60/HSP10 complex was evaluated by a trypsin sensitivity assay using heat-denatured rhodanese in the presence or absence of ATP, ATP-AlFx (C), or GTP and GTP-AlFx (D). After initiation of the refolding reaction, samples were taken at 1 min or 60 min and treated with 10 μg/ml trypsin for 3 min. Samples were separated by 12% SDS-PAGE and the protein bands were detected by Coomassie Brilliant Blue R250-staining. The asterisk indicates the proteolytic fragment of the folded rhodanese digested by trypsin. The area of native rhodanese proteolytic fragments of the upper panels gels (the portion enclosed with a red frame) was analyzed under the high-resolution conditions (as shown in lower panels C and D). It is noted that heat denaturation has no effect on the function of HSP60 (see also supplementary Figure S3).

Figure 5

Enzymatic activity of HSP60 in the ATPase –dependent manner in the presence of GTP. (A), ATPase activity of HSP60 with or without HSP10 in the presence or absence of GTP. The NTP hydrolysis assay was performed in the presence of various concentrations of ATP with or without 0.5 mM GTP for 60 min. (B,C), Rhodanese refolding activity of HSP60/HSP10 in the presence of various concentrations of ATP with or without GTP. The refolding reaction was performed in the presence of 0.01, 0.5, 1.0 or 3.5 mM ATP with or without 0.5 mM GTP. The refolding yield was calculated by measuring the recovery of the enzymatic activity of rhodanese.

Figure 6

In silico modeling of the HSP60 binding ATP or GTP. (A) The 3D modeling of the HSP60 with ATP. (B) The 3D modeling of the HSP60 with GTP. The docking simulation was performed using MF myPresto v2 (FiatLux). The green boxes in the enlarged views showed the Asp398. The ribbon model shows the HSP60 (PDB entry is 4PJ1) and the ball models show the ATP and GTP (PDB entries are 1Q12 and 2YWQ, respectively).