Identification of Elements That Dictate the Specificity of Mitochondrial Hsp60 for Its Co-Chaperonin

Abstract

Type I chaperonins (cpn60/Hsp60) are essential proteins that mediate the folding of proteins in bacteria, chloroplast and mitochondria. Despite the high sequence homology among chaperonins, the mitochondrial chaperonin system has developed unique properties that distinguish it from the widely-studied bacterial system (GroEL and GroES). The most relevant difference to this study is that mitochondrial chaperonins are able to refold denatured proteins only with the assistance of the mitochondrial co-chaperonin. This is in contrast to the bacterial chaperonin, which is able to function with the help of co-chaperonin from any source. The goal of our work was to determine structural elements that govern the specificity between chaperonin and co-chaperonin pairs using mitochondrial Hsp60 as model system. We used a mutagenesis approach to obtain human mitochondrial Hsp60 mutants that are able to function with the bacterial co-chaperonin, GroES. We isolated two mutants, a single mutant (E321K) and a double mutant (R264K/E358K) that, together with GroES, were able to rescue an E. coli strain, in which the endogenous chaperonin system was silenced. Although the mutations are located in the apical domain of the chaperonin, where the interaction with co-chaperonin takes place, none of the residues are located in positions that are directly responsible for co-chaperonin binding. Moreover, while both mutants were able to function with GroES, they showed distinct functional and structural properties. Our results indicate that the phenotype of the E321K mutant is caused mainly by a profound increase in the binding affinity to all co-chaperonins, while the phenotype of R264K/E358K is caused by a slight increase in affinity toward co-chaperonins that is accompanied by an alteration in the allosteric signal transmitted upon nucleotide binding. The latter changes lead to a great increase in affinity for GroES, with only a minor increase in affinity toward the mammalian mitochondrial co-chaperonin.

Crystal structure of the human mitochondrial chaperonin symmetrical football complex

“…Two observations support our assumption that the structure determined here likely represents an event in the cycle just before mHsp10 dissociates from mHsp60: (i) Surface plasmon resonance measurements showed that the association step of mHsp60 E321K with mHsp10 is similar to the association step of the WT Hsp60, whereas in the case of the mutant, the dissociation step is markedly prolonged (31), and (ii) despite the fact that the complex was crystallized in the presence of ATP, the nucleotide occupying the binding site in the football structure is ADP in all of the subunits (as will be discussed below).…”

“…This lack of functionality was shown to be due to its inability to release mHsp10 (detailed in ref. 31). Further experiments with the mHsp60 E321K mutant using the substrate EGFP revealed that the mutant is able to functionally encapsulate and refold HCl-denatured EGFP and to release it within the chaperonin cavity.…”

“…Thus, the mobile loops of mHsp10 subunits U and 2 (the nomenclature is provided in Fig. S4) evidently initiate the dissociation move but do not proceed further, owing to stabilization of the open state of mHsp60 E321K (31). We can speculate that complete dissociation of the mobile loop from mHsp60 is gradual, and occurs once the apical domain begins the downward movement toward its apo conformation.…”

“…crystallization of this complex, we eliminated the salt bridge between E321 and K176 (mHsp60 E321K ) that is known to break upon transition from the closed conformation to the open conformation 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).…”

Alzheimer's Disease and Molecular Chaperones: Current Knowledge and the Future of Chaperonotherapy

“…Inside mitochondria, Hsp60 acts as a folding machine, together with Hsp10, for the correct folding of several mitochondrial proteins [40,41]. Various studies, published over the past several years, have demonstrated new subcellular locations and functions for Hsp60, describing it as a ubiquitous molecule with multiple roles in health and disease [42][43][44].…”