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

Type I chaperonins: not all are created equal

AbstractType I chaperonins play an essential role in the folding of newly translated and stress-denatured proteins in eubacteria, mitochondria and chloroplasts. Since their discovery, the bacterial chaperonins have provided an excellent model system for investigating the mechanism by which chaperonins mediate protein folding. Due to the high conservation of the primary sequence among Type I chaperonins, it is generally accepted that organellar chaperonins function similar to the bacterial ones. However, recent studies indicate that the chloroplast and mitochondrial chaperonins possess unique structural and functional properties that distinguish them from their bacterial homologs. This review focuses on the unique properties of organellar chaperonins

The inhibitory effect of ADP on MDH refolding activity by chaperonins.

<p>(A–D) Refolding of 0.33 µM HCl-denatured MDH by 10 µM of the indicated chaperonin and 40 µM of mHsp10 (white columns) or GroES (black columns). MDH activity was measured at 340 nm following a 60 min incubation at 30°C in the absence of nucleotides or in the presence of 10 mM ADP, 1 mM ATP or 1 mM ATP+10 mM ADP as indicated. The activity following refolding is presented relative to that of native MDH (100%). (E–G) Time-dependent refolding activity of wild-type mHsp60 (E), E321K mutant (F) and R264K/E358K mutant (G) together with mHsp10 (white symbols) or GroES (black symbols) in the presence of 1 mM ATP (triangles) or 1 mM ATP+10 mM ADP (squares). The relative activity is compared to the activity measured by each chaperonin pair after 30 min in the presence of ATP (100%).</p

Chaperonin-co-chaperonin interactions measured by SPR.

<p>Association and dissociation patterns of 10 µM of the indicated chaperonin to immobilized (A) GroES (∼ 600 Relative Units-RU) or (B) mHsp10 (∼ 800 RU) in the presence of 2 mM ATP.</p

SPR analysis.

1<p>Ratios of association equilibrium constant represent the apparent K_A measured between each pair relative to the apparent K_A measured between mHsp60 and mHsp10 (apparent K_D of 7.4 µM). The apparent values of K_A, the association constant (M⁻¹), were determined using equilibrium analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050318#pone.0050318-GevorkyanAirapetov1" target="_blank">[65]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050318#pone.0050318-Marom1" target="_blank">[66]</a>. Values represent average ± SEM of at least three independent experiments.</p>2<p>ND, no binding detected.</p

Identifying mHsp60 mutants that are functional with GroES.

<p>(A) Examination of the <i>in vivo</i> system at the indicated growth conditions. (B) Ten-fold-serial dilutions of <i>E. coli</i> strain MGM100 harboring plasmid pOFX with the indicated mHsp60 variant and GroES, grown on agar plates in the presence of glucose and IPTG. (C–D) Examination of the ability of mHsp60 mutants to facilitate the growth of MGM100 on agar plates containing glucose and IPTG in combination with GroES (C) or mHsp10 (D). GroEL-GroES and mHsp60-mHsp10 combinations serve as positive controls; the mHsp60-GroES combination serves as negative control. (E) Refolding of 0.33 µM HCl-denatured MDH by 10 µM of the indicated chaperonin and 40 µM of mHsp10 (white columns) or GroES (black columns). MDH activity was measured at 340 nm following 120 min incubation at 30°C in the presence of 1 mM ATP. The activity following refolding is presented relative to that of native MDH (100%).</p

The effect of the K176E mutation on the function of mHsp60 with co-chaperonins.

<p>Refolding of 0.33 µM HCl-denatured MDH by 10 µM of the indicated chaperonin and 20 µM of mHsp10 (white columns) or GroES (black columns). MDH activity was measured at 340 nm following 60 min incubation at 30 °C in the presence of 1 mM ATP. The activity following refolding is presented relative to that of native MDH (100%).</p

A stable complex is formed between the E321K mutant and mHsp10.

<p>Interaction between mHsp10 and different chaperonins was measured using a pulldown assay. 50 µM of His-tagged mHsp10 together with 50 µM of GroEL (A), mHsp60 (B), or E321K mutant (C) were incubated with nickel beads in the absence of nucleotides, or in the presence of 4 mM ATP or 4 mM ADP. Equivalent aliquots of 2 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 blue. The intensities of the bands were quantified by densitometry (ImageMaster 1D Prime program). The bound ratio listed on the bottom of each gel represents the ratio between the intensities of the chaperonin and co-chaperonin bands in the bound fraction.</p

Positions of mutations in the primary and tertiary structures of GroEL.

<p>(A) Alignment of the amino acid sequences of GroEL and the mature mHsp60 protein. Protein sequence alignments were carried out by ClustalW. Amino acids discussed in this study are marked in boldface type. The amino acids known to be in direct contact with GroES are underlined. The color code corresponding to domain boundaries is described below. (B–C) 3D-structure models of GroEL subunit in the down (B) and up (C) conformations (Protein Data Bank entry 1AON) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050318#pone.0050318-Xu1" target="_blank">[43]</a>. The amino acids discussed in this study are labeled and presented as space-filling models. The corresponding amino acids in mHsp60 are indicated in brackets. The ADP molecule is colored in purple. The three domains as defined by GroEL are color-coded on the GroEL sequence and structure: equatorial (blue), intermediate (green) and apical (red). Helices H and I are colored in gray. The figure was produced using PyMOL software.</p

Inhibition of GroES-E321K refolding activity by mHsp10.

<p>A binary complex of E321K and HCl-denaturated MDH was pre-incubated for 30 min in the presence of increasing concentrations (from 0 to 20 µM) of mHsp10 and 2 mM ATP before adding 20 µM GroES. MDH activity was measured 1 hour following the addition of GroES. % inhibition = 100*[(A_o–A_i)/A_o]. A_o represents the activity level in the absence of mHsp10, and A_i represents the activity level at each mHsp10 concentration.</p