Crystal structure of the human mitochondrial chaperonin symmetrical football complex Shahar Nisemblata,b, Oren Yanivb,c, Avital Parnasa,b, Felix Frolowb,c,1, and Abdussalam Azema,b,2 Departments of aBiochemistry and Molecular Biology and cMolecular Microbiology and Biotechnology, and bThe Daniella Rich Institute for Structural Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel Edited* by George H. Lorimer, University of Maryland, College Park, MD, and approved April 1, 2015 (received for review June 22, 2014) Human mitochondria harbor a single type I chaperonin system that via a mechanism distinct from the mechanism of GroEL/GroES is generally thought to function via a unique single-ring interme- and acts as a single heptameric ring (16–18). This chaperonin diate. To date, no crystal structure has been published for any system is crucial for mitochondrial function and cellular viability, mammalian type I chaperonin complex. In this study, we describe as demonstrated by the embryonic lethality in mice that results the crystal structure of a football-shaped, double-ring human mito- from inactivation of the mHsp60 gene, as well as by the identi- chondrial chaperonin complex at 3.15 Å, which is a novel inter- fication of three human neurodegenerative genetic disorders mediate, likely representing the complex in an early stage of associated with mutations in this protein (19–23). In addition dissociation. Interestingly, the mitochondrial chaperonin was cap- to their essential protein-folding activity in mitochondria, tured in a state that exhibits subunit asymmetry within the rings studies have implicated the mammalian mitochondrial chap- and nucleotide symmetry between the rings. Moreover, the chap- eronins in a wide range of other extramitochondrial and extra- eronin tetradecamers show a different interring subunit arrange- cellular activities, including modulation of apoptosis (24, 25), ment when compared to GroEL. Our findings suggest that the inflammation (26), and carcinogenesis (reviewed in refs. 27–30). mitochondrial chaperonins use a mechanism that is distinct from Here we report, the first crystal structure of mHsp60 in Escherichia coli the mechanism of the well-studied system. complex with its cochaperonin. The model that we obtained provides a snapshot of a unique intermediate, likely in a step mitochondrial chaperonin | symmetrical complex | Hsp60 | chaperone | preceding the dissociation of the complex into its components, Hsp10 that exhibits three unique properties: (i) the mHsp60–mHsp10 complex forms a symmetric double-ring, football-like structure he type I chaperonin family is essential for the life of all [mHsp6014–(mHsp107)2 complex] that displays extensive in- Teukaryotes, because the main function of its members is to terring contacts; (ii) the symmetric nature of the chaperonin mediate actively the folding of cellular proteins (1–4). The bacterial subunits within each ring, which is observed in GroEL, is not chaperonin GroEL/GroES system is the most intensively studied preserved; and (iii) the interring nucleotide asymmetry that de- chaperonin system, owing to its profound stability, and it serves as a fines the bacterial folding cycle is absent, because both mHsp60 prototype for the less stable 60-kDa chaperonins from mitochon- rings are in the ADP-bound state. dria and chloroplasts. The GroEL molecule is a tetradecamer whose subunits are organized in two rings, producing a barrel-like Results structure. The central cavity of the barrel provides an isolated Structure Determination and Refinement. The mHsp60–mHsp10 environment for protein folding, a process that requires the assis- complex is known for its intrinsic instability (16, 17). To facilitate tance of the cochaperonin GroES as well as ATP hydrolysis (5, 6). Several intermediate complexes were suggested to be key Significance players in the GroEL/GroES reaction cycle. The transition be- tween the various intermediates is governed by the state of the The human mitochondrial chaperonin is vital for proper cell nucleotide molecules bound to GroEL. In vitro, the acceptor function because it assists in folding of mitochondrial proteins. state for the unfolded protein substrate is either the apo or the – Additionally, it participates in extramitochondrial processes, asymmetric form of the GroEL GroES complex (a bullet-shaped such as apoptosis, inflammation, and carcinogenesis. In this complex containing one molecule of GroEL and one molecule of study, we report the crystal structure of mitochondrial chap- GroES). The protein-folding cycle begins when GroES binds to “ ” cis eronins. The model shows an American football -shaped in- the ring occupied by substrate protein and ATP (the ring). termediate, composed of two 7-membered chaperonin rings ATP hydrolysis in the cis ring is followed by binding of ATP to trans cis capped at each end by a cochaperonin ring. This complex was the ring, which results in dissociation of the complex, captured in the early stages of dissociation. The extensive in- thereby releasing the folded substrate protein and resetting the terface contacts between its rings, the asymmetry that exists – cycle (7 9). However, numerous studies have demonstrated that within each ring, and symmetric binding of nucleotide cofactors an additional complex is formed between GroEL and GroES that exists between the rings suggest that the mitochondrial during the protein-folding reaction cycle. This symmetric com- system operates via a mechanism that is distinct from the mech- “ ” plex, called the football (American football-shaped), was shown anism of the canonical Escherichia coli (GroEL/GroES) system. to be a functional intermediate of the chaperonin folding reaction cycle (10–13). Of all the GroEL/GroES chaperonin intermediates, Author contributions: S.N., A.P., F.F., and A.A. designed research; S.N. and A.P. performed the symmetric complex is the only form whose structure has not research; S.N., O.Y., and F.F. analyzed data; and S.N., F.F., and A.A. wrote the paper. been determined until recently (published during the review of this The authors declare no conflict of interest. paper), apparently because of its transient nature (14, 15). *This Direct Submission article had a prearranged editor. Human mitochondria harbor one chaperonin system whose Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, proteins, human mitochondrial heat shock protein 60 (mHsp60) www.pdb.org (PDB ID code 4PJ1). and its cochaperonin, human mitochondrial heat shock protein 10 1Deceased August 28, 2014. (mHsp10), exhibit 51% and 33% identity to GroEL and GroES, 2To whom correspondence should be addressed. Email: [email protected]. respectively. The accepted model of the protein-folding reaction This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cycle by mHsp60 suggests that this folding nanomachine functions 1073/pnas.1411718112/-/DCSupplemental. 6044–6049 | PNAS | May 12, 2015 | vol. 112 | no. 19 www.pnas.org/cgi/doi/10.1073/pnas.1411718112 Downloaded by guest on September 29, 2021 crystallization of this complex, we eliminated the salt bridge between E321 and K176 (mHsp60E321K) that is known to break upon transition from the closed conformation to the open con- formation during the reaction cycle, leading to the formation of a high-affinity complex. We previously isolated this mutant and showed that it has acquired the ability to function with GroES both in vivo and in vitro, although losing the ability to function with its endogenous partner, mHsp10 (31) (Fig. S1). This lack of functionality was shown to be due to its inability to release mHsp10 (detailed in ref. 31). Further experiments with the mHsp60E321K mutant using the substrate EGFP revealed that the mutant is able to functionally encapsulate and refold HCl-dena- tured EGFP and to release it within the chaperonin cavity. The mutant has only lost the ability to open the cavity and to discharge the folded protein (Fig. S2). This method was used previously to characterize the SR1 mutant of GroEL, which is arrested in the same stage as our mutant, as described by Weissman et al. (32). These results indicate that the stable mHsp60–mHsp10 complex represents a true intermediate of the mHsp60 folding cycle. Crystallization trials were carried out with purified mHsp60E321K lacking the C-terminal GGM motif. Removing the GGM tail, Fig. 1. Overview of the mHsp60–mHsp10 football complex. (A) View per- which is predicted to be unstructured, improved data resolution. pendicular to the long axis of the complex. The seven subunits in each Crystallization of the mHsp60E321K mutant in the presence of mHsp60 ring are colored red, orange, yellow, green, cyan, blue, and purple. The mHsp10 subunits in each ring are colored blue, light blue, purple, dark mHsp10 turned out to be advantageous on two counts: (i) The E321K– purple, yellow, pink, and dark red. Structure dimensions are marked by ar- high stability of the mHsp60 mHsp10 complex helped to rows (distances between Cα). Atoms of the 14 ADP molecules are rendered overcome the labile nature of the mHsp60 molecule, and (ii) as spheres. (B) Views from the north and south poles of the mHsp60 – “ ” 14 the E321K mutation stabilizes the open conformation of the (mHsp107)2 complex. The coloring scheme is as in A. molecule, making it possible to obtain crystals of mHsp60 that are fully complexed with mHsp10. mHsp60E321K and His-tagged mHsp10 were cocrystallized in