The human mitochondrial Hsp60 in the APO conformation forms a stable tetradecameric complex

ABSTRACT

The human mitochondrial chaperonin is a macromolecular machine that catalyzes the proper folding of mitochondrial proteins and is of vital importance to all cells. This chaperonin is composed of 2 distinct proteins, Hsp60 and Hsp10, that assemble into large oligomeric complexes that mediate the folding of non-native polypeptides in an ATP dependent manner. Here, we report the bacterial expression and purification of fully assembled human Hsp60 and Hsp10 recombinant proteins and that Hsp60 forms a stable tetradecameric double-ring conformation in the absence of co-chaperonin and nucleotide. Evidence of the stable double-ring conformation is illustrated by the 15 Å resolution electron microscopy reconstruction presented here. Furthermore, our biochemical analyses reveal that the presence of a non-native substrate initiates ATP-hydrolysis within the Hsp60/10 chaperonin to commence protein folding. Collectively, these data provide insight into the architecture of the intermediates used by the human mitochondrial chaperonin along its protein folding pathway and lay a foundation for subsequent high resolution structural investigations into the conformational changes of the mitochondrial chaperonin.

KEYWORDS:

• Human mitochondrial chaperonin
• negative stain
• transmission electron microscopy

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We would like to thank Dr. Chuan Xiao and Jennie Choi for their assistance with the DLS experiments.

Funding

This work was made possible by the Welch Foundation award (AH-1649) and NIH-NIGMS (SC3GM113805) awarded to Ricardo A. Bernal. This work was supported by Grant 5G12MD007592 from the National Institutes on Minority Health and Health Disparities (NIMHD), a component of the National Institutes of Health (NIH).