Identification of elements that dictate the specificity of mitochondrial Hsp60 for its co-chaperonin

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

Dynamic complexes in the chaperonin-mediated protein folding cycle

The GroEL-GroES chaperonin system is probably one of the most studied chaperone systems at the level of the molecular mechanism. Since the first reports of a bacterial gene involved in phage morphogenesis in 1972, these proteins have stimulated intensive research for over 40 years. During this time, detailed structural and functional studies have yielded constantly evolving concepts of the chaperonin mechanism of action. Despite of almost three decades of research on this oligomeric protein, certain aspects of its function remain controversial. In this review, we highlight one central aspect of its function, namely, the active intermediates of its reaction cycle, and present how research to this day continues to change our understanding of chaperonin-mediated protein folding

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

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.We acknowledge support from the United States-Israel Binational Science Foundation (grant number 2015214) to A.A. and I.U.-B. F.J. was supported by a fellowship from the Planning and Budgeting Committee of the Israel Council for Higher Education. I.U.-B. was supported in part by the Fundación Biofísica Bizkaia and the Basque Excellence Research Centre program.Peer reviewe

<em>P. falciparum</em> cpn20 Is a Bona Fide Co-Chaperonin That Can Replace GroES in <em>E. coli</em>

<div><p>Human malaria is among the most ubiquitous and destructive tropical, parasitic diseases in the world today. The causative agent, <em>Plasmodium falciparum</em>, contains an unusual, essential organelle known as the apicoplast. Inhibition of this degenerate chloroplast results in second generation death of the parasite and is the mechanism by which antibiotics function in treating malaria. In order to better understand the biochemistry of this organelle, we have cloned a putative, 20 kDa, co-chaperonin protein, Pf-cpn20, which localizes to the apicoplast. Although this protein is homologous to the cpn20 that is found in plant chloroplasts, its ability to function as a co-chaperonin was questioned in the past. In the present study, we carried out a structural analysis of Pf-cpn20 using circular dichroism and analytical ultracentrifugation and then used two different approaches to investigate the ability of this protein to function as a co-chaperonin. In the first approach, we purified recombinant Pf-cpn20 and tested its ability to act as a co-chaperonin for GroEL <em>in vitro</em>, while in the second, we examined the ability of Pf-cpn20 to complement an <em>E. coli</em> depletion of the essential bacterial co-chaperonin GroES. Our results demonstrate that Pf-cpn20 is fully functional as a co-chaperonin <em>in vitro</em>. Moreover, the parasitic co-chaperonin is able to replace GroES in <em>E. coli</em> at both normal and heat-shock temperatures. Thus, Pf-cpn20 functions as a co-chaperonin in chaperonin-mediated protein folding. The ability of the malarial protein to function in <em>E. coli</em> suggests that this simple system can be used as a tool for further analyses of Pf-cpn20 and perhaps other chaperone proteins from <em>P. falciparum</em>.</p> </div

Pf-cpn20 successfully replaces the function of GroES in <i>E. coli</i>.

<p> Complementation assays were carried out in a strain of <i>E. coli</i>, MGM100, in which expression of endogenous chaperonins (GroEL-GroES) was under strict control of the pBAD promoter. (A) Various controls for the <i>in vivo</i> system at the indicated growth conditions (10⁻² dilution shown). (B and C) Ten-fold-serial dilutions (10⁻² to 10⁻⁷ shown) of <i>E. coli</i> strain MGM100 harboring plasmid pOFX containing GroEL and the indicated co-chaperonin, grown on agar plates in the presence of glucose and IPTG, but no arabinose: B) at 30°C C) at 44°C.</p

Refolding of denatured MDH by GroEL and Pf-cpn20.

<p>Urea-denatured malate dehydrogenase was refolded by GroEL with the help of Pf-cpn20, using At-cpn20 and GroES as control co-chaperonins. A) Refolding yields as a function of the co-chaperonin/GroEL protomer ratio. The refolding reaction was carried out for 30 minutes with 10 µM GroEL and increasing co-chaperonin concentration (0.16 to 20 µM), as described in Materials and Methods. Refolding is expressed relative to the highest yield obtained with GroES. B) Refolding yield as a function of time carried out with 10 µM GroEL and a sub-saturating co-chaperonin concentration (2 µM) Values represent the average of 3 independent experiments +/− standard deviation.</p

Analytical ultracentrifugation values for GroES, At-cpn20 and Pf-cpn20.

*<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053909#pone.0053909-Applied1" target="_blank">[46]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053909#pone.0053909-Gur1" target="_blank">[48]</a>.</p

Pf-cpn20 does not form hetero-oligomers with At-cpn10.

<p>Interaction between Cpn20 and Cpn10 was measured using a pulldown assay. His-tagged At- or Pf-cpn20 was incubated with At-cpn10 and bound to Ni²⁺ beads as described in Materials and Methods. Equivalent aliquots of 12 µl from the total sample (T), unbound fraction (U), fourth wash (W), and bound fraction (B) were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue R-250.</p

Deconvolution of CD data for At-cpn20 and Pf-cpn20^*.

*<p>Carried out on 1 mg/ml protein, 190–260 nm using the CDNN program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053909#pone.0053909-deJongh1" target="_blank">[42]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053909#pone.0053909-Fossati1" target="_blank">[43]</a>.</p