Human mitochondrial chaperonin mHsp60 is broadly associated with various human health conditions and the V72I mutation in mHsp60 causes a form of hereditary spastic paraplegia, a neurodegenerative disease. The main function of mHsp60 is to assist folding of mitochondrial proteins in an ATP-dependent manner. In this study, we unexpectedly found that mutant mHsp60V72I was more stable structurally and more active in the ATPase activity than the wildtype. Analysis of our recently solved cryo-EM structure of mHsp60 revealed allosteric roles of V72I in structural stability and ATPase activity, which were supported by studies including those using the V72A mutation. Despite with the increases in structural stability and ATPase activity, mHsp60V72I was less efficient in folding malate dehydrogenase, a putative mHsp60 substrate protein in mitochondria and also commonly used in chaperonin studies. In addition, although mHsp60V72I along with its cochaperonin mHsp10 was able to substitute the E. coli chaperonin system in supporting cell growth under normal temperature of 37 °C, it was unable under heat shock temperature of 42 °C. Our results support the importance of structural dynamics and an optimal ATP turnover that mHsp60 has evolved for its function and physiology. We propose that unproductive energy utilization, or hyperactive ATPase activity and compromised folding function, not mutually exclusive, are responsible for the V72I pathology in neurodegenerative disease.

The human mitochondrial heat shock protein 60 (hsp60) is a tetradecameric chaperonin that folds proteins in the mitochondrial matrix. An hsp60 D3G mutation leads to MitCHAP-60, an early onset neurodegenerative disease while hsp60 V72I has been linked to SPG13, a form of hereditary spastic paraplegia. Previous studies have suggested that these mutations impair the protein folding activity of hsp60 complexes but the detailed mechanism by which these mutations lead the neuromuscular diseases remains unknown. It is known, is that the β-subunit of the human mitochondrial ATP synthase co-immunoprecipitates with hsp60 indicating that the β-subunit is likely a substrate for the chaperonin. Therefore, we hypothesized that hsp60 mutations cause misfolding of proteins that are critical for aerobic respiration. Negative-stain electron microscopy and DLS results suggest that the D3G and V72I complexes fall apart when treated with ATP or ADP and are therefore unable to fold denatured substrates such as α-lactalbumin, malate dehydrogenase (MDH), and the β-subunit of ATP synthase in in-vitro protein-folding assays. These data suggests that hsp60 plays a crucial role in folding important players in aerobic respiration such as the β-subunit of the ATP synthase. The hsp60 mutations D3G and V72I impair its ability to fold mitochondrial substrates leading to abnormal ATP synthesis and the development of the MitCHAP-60 and SPG13 neuromuscular degenerative disorders.