Structural basis for active single and double ring complexes in human mitochondrial Hsp60-Hsp10 chaperonin

Abstract

3,4 ✉ 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:BeF 3 -bound) ground-state mimic double-ring mHsp60 14 -(mHsp10 7 ) 2 football complex, and the cryo-EM structures of the ADP-bound successor mHsp60 14 -(mHsp10 7 ) 2 complex, and a single-ring mHsp60 7 -mHsp10 7 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 halffootballs. 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.

Missense Mutations of Human Hsp60: A Computational Analysis to Unveil Their Pathological Significance

“…Also S488, corresponding to S462 in mHsp60, which has been recently reported to be involved in inter-ring contacts (Gomez-Llorente et al, 2020), was found to be replaced by a threonine ( Table 1 and Supplementary Table S1). The important role of this amino acid suggests a potential deleterious effect of its substitution.…”

“…The residues Asp504, Ala505, and Met506 are located in the nucleotide-binding pocket of hHsp60, as inferred from the solved crystal structures (Nisemblat et al, 2015;Gomez-Llorente et al, 2020). The corresponding amino acids in GroEL, i.e., Asn479, Ala480, and Ala481, involved in nucleotide binding (Boisvert et al, 1996;Xu et al, 1997;Brocchieri and Karlin, 2000), have different identities, but maintain the same location in proteins three-dimensional structures (Figures 1, 2G-I).…”

“…Glu129 and Lys133 of hHsp60 full-length, corresponding to Glu103 and Lys107 in mHsp60, are linked by a salt bridge (Nisemblat et al, 2015;Gomez-Llorente et al, 2020). Both these residues were found replaced by a lysine and a glutamic acid, respectively ( Table 1 and Supplementary Table S1), and both these replacements could prevent the formation of the described salt bridge, altering the stability of the tetradecamer.…”

“…Met55 sits in the nucleotide binding pocket, as inferred from the analysis of the recent solved crystal structure of the ADP:BeF3-bound (ATP ground state) wild-type mHsp60 14 -(mHsp10 7 ) 2 football complex (Gomez-Llorente et al, 2020). Met55 corresponds to GroEL Leu31 that is known to be involved in nucleotide binding (Xu et al, 1997;Brocchieri and Karlin, 2000;Wang and Boisvert, 2003).…”

“…Moreover, in solution it exists in dynamic equilibrium between monomers, heptameric single rings and double-ring tetradecamers, and only the presence of both ATP and Hsp10 favors the formation of the functional football complex (Levy-Rimler et al, 2001). Not yet fully elucidated is also the mechanism of action, even if it likely involves different intermediates, including the ADP-bound half football complex and the ATP and ADP-bound football complexes (Gomez-Llorente et al, 2020).…”