Significance: The human mitochondrial chaperonin is vital for proper cell function because it assists in folding of mitochondrial proteins. Additionally, it participates in extramitochondrial processes, such as apoptosis, inflammation, and carcinogenesis. In this study, we report the crystal structure of mitochondrial chaperonins. The model shows an “American football”-shaped intermediate, composed of two 7-membered chaperonin rings capped at each end by a cochaperonin ring. This complex was captured in the early stages of dissociation. The extensive interface contacts between its rings, the asymmetry that exists within each ring, and symmetric binding of nucleotide cofactors that exists between the rings suggest that the mitochondrial system operates via a mechanism that is distinct from the mechanism of the canonical Escherichia coli (GroEL/GroES) system.

MHsp60-mHsp10 assists the folding of mitochondrial matrix proteins without the negative ATP binding inter-ring cooperativity of GroEL-GroES. Here we report the crystal structure of an ATP (ADP:BeF₃-bound) ground-state mimic double-ring mHsp60₁₄-(mHsp10₇)₂ football complex, and the cryo-EM structures of the ADP-bound successor mHsp60₁₄-(mHsp10₇)₂ complex, and a single-ring mHsp60₇-mHsp10₇ half-football. The structures explain the nucleotide dependence of mHsp60 ring formation, and reveal an inter-ring nucleotide symmetry consistent with the absence of negative cooperativity. In the ground-state a two-fold symmetric H-bond and a salt bridge stitch the double-rings together, whereas only the H-bond remains as the equatorial gap increases in an ADP football poised to split into half-footballs. Refolding assays demonstrate obligate single- and double-ring mHsp60 variants are active, and complementation analysis in bacteria shows the single-ring variant is as efficient as wild-type mHsp60. Our work provides a structural basis for active single- and double-ring complexes coexisting in the mHsp60-mHsp10 chaperonin reaction cycle.

Precursor proteins that are imported from the cytosol into the matrix of mitochondria carry positively charged amphipathic presequences and cross the inner membrane with the help of vital components of the TIM23 complex. It is currently unclear which subunits of the TIM23 complex recognize and directly bind to presequences. Here we analyzed the binding of presequence peptides to purified components of the TIM23 complex. The interaction of three different presequences with purified soluble domains of yeast Tim50 (Tim50IMS), Tim23 (Tim23IMS), and full-length Tim44 was examined. Using chemical cross-linking and surface plasmon resonance we demonstrate, for the first time, the ability of purified Tim50IMS and Tim44 to interact directly with the yeast Hsp60 presequence. We also analyzed their interaction with presequences derived from precursors of yeast mitochondrial 70-kDa heat shock protein (mHsp70) and of bovine cytochrome P450SCC. Moreover, we characterized the nature of the interactions and determined their KDs. On the basis of our results, we suggest a mechanism of translocation where stronger interactions of the presequences on the trans side of the channel support the import of precursor proteins through TIM23 into the matrix.

The majority of mitochondrial proteins use N-terminal presequences for targeting to mitochondria and are translocated by the presequence translocase. During translocation, proteins, threaded through the channel in the inner membrane, are handed over to the import motor at the matrix face. Tim17 is an essential, membrane-embedded subunit of the translocase; however, its function is only poorly understood. Here, we functionally dissected its four predicted transmembrane (TM) segments. Mutations in TM1 and TM2 impaired the interaction of Tim17 with Tim23, component of the translocation channel, whereas mutations in TM3 compromised binding of the import motor. We identified residues in the matrix-facing region of Tim17 involved in binding of the import motor. Our results reveal functionally distinct roles of different regions of Tim17 and suggest how they may be involved in handing over the proteins, during their translocation into mitochondria, from the channel to the import motor of the presequence translocase.

The 60-kDa heat shock protein (mHsp60) is a vital cellular complex that mediates the folding of many of the mitochondrial proteins. Its function is executed in cooperation with the co-chaperonin, mHsp10, and requires ATP. Recently, the discovery of a new mHsp60-associated neurodegenerative disorder, MitCHAP-60 disease, has been reported. The disease is caused by a point mutation at position 3 (D3G) of the mature mitochondrial Hsp60 protein, which renders it unable to complement the deletion of the homologous bacterial protein in Escherichia coli (Magen, D., Georgopoulos, C., Bross, P., Ang, D., Segev, Y., Goldsher, D., Nemirovski, A., Shahar, E., Ravid, S., Luder, A., Heno, B., Gershoni-Baruch, R., Skorecki, K., and Mandel, H. (2008) Am. J. Hum. Genet. 83, 30-42). The molecular basis of the MitCHAP-60 disease is still unknown. In this study, we present an in vitro structural and functional analysis of the purified wild-type human mHsp60 and the MitCHAP-60 mutant. We show that the D3G mutation leads to destabilization of the mHsp60 oligomer and causes its disassembly at low protein concentrations. We also show that the mutant protein has impaired protein folding and ATPase activities. An additional mutant that lacks the first three amino acids (N-del), including Asp-3, is similarly impaired in refolding activity. Surprisingly, however, this mutant exhibits profound stabilization of its oligomeric structure. These results suggest that the D3G mutation leads to entropic destabilization of the mHsp60 oligomer, which severely impairs its chaperone function, thereby causing the disease.