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. 2010 Dec 1;29(23):4008-19.
doi: 10.1038/emboj.2010.262. Epub 2010 Oct 19.

Polypeptide in the chaperonin cage partly protrudes out and then folds inside or escapes outside

Fumihiro Motojima  1 , Masasuke Yoshida
Affiliations

Polypeptide in the chaperonin cage partly protrudes out and then folds inside or escapes outside

Fumihiro Motojima et al. EMBO J. .

Abstract

The current mechanistic model of chaperonin-assisted protein folding assumes that the substrate protein in the cage, formed by GroEL central cavity capped with GroES, is isolated from outside and exists as a free polypeptide. However, using ATPase-deficient GroEL mutants that keep GroES bound, we found that, in the rate-limiting intermediate of a chaperonin reaction, the unfolded polypeptide in the cage partly protrudes through a narrow space near the GroEL/GroES interface. Then, the entire polypeptide is released either into the cage or to the outside medium. The former adopts a native structure very rapidly and the latter undergoes spontaneous folding. Partition of the in-cage folding and the escape varies among substrate proteins and is affected by hydrophobic interaction between the polypeptide and GroEL cavity wall. The ATPase-active GroEL with decreased in-cage folding produced less of a native model substrate protein in Escherichia coli cells. Thus, the polypeptide in the critical GroEL-GroES complex is neither free nor completely confined in the cage, but it is interacting with GroEL's apical region, partly protruding to outside.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Rhodanese in the SR398–GroES cage is accessible from outside. (A, B) Interference with SR398-assisted folding of rhodanese (0.1 μM) by 4 μM anti-rhodanese antibody (A) or 5 μM trap(N265A) (B) added at the time indicated by arrows. Rhodanese activity with and without antibody or trap(N265A) is shown by open and closed circles, respectively. (C) Stable association of the SR398–GroES complex in the presence of antibody, trap(N265A) and trap(D87K). Exchange of fluorescently labelled GroESAEDANS associated with SR398 by non-labelled GroES in the medium was examined. The SR398–GroESAEDANS–rhodanese ternary complex was formed by addition of ATP. Antibody, trap(N265A) or trap(D87K) was added at the same time. Subsequently, a 10-fold molar excess of non-labelled GroES was mixed. After a 30-min incubation, the solutions were applied to a gel-filtration column and the fluorescence of AEDANS was monitored. Percent values of GroESAEDANS associated with SR398 are shown. (D) Conjugate formation of rhodanese with anti-rhodanese antibody and trap(N265A) during the SR398-assisted folding. The result of trap(D87K), which captured only free rhodanese, is also shown. Fluorescently labelled rhodaneseAlexa was used as a substrate protein. The SR398–GroES–rhodaneseAlexa ternary complex was formed by addition of ATP together with antibody, trap(N265A) or trap(D87K) and, after a 30-min incubation, the solutions were applied to a gel-filtration column with monitoring of the fluorescence of Alexa. The concentrations of anti-rhodanese antibody, trap(N265A) and trap(D87K) were 4, 5 and 0.2 μM, respectively. (E) Chymotrypsin digestion of rhodanese during the SR398-assisted folding. At the indicated time, an aliquot was incubated with chymotrypsin for 5 min and the samples were analysed by SDS–PAGE (inset). The intensity of the stained protein bands of undigested rhodanese relative to that without chymotrypsin treatment (none) was plotted. Details are described in Materials and methods.
Figure 2
Figure 2
Natural and forced escape of rhodanese during the SR398-assisted folding. (A) Time course of the SR398-assisted folding of rhodanese in the presence of three different concentrations of trapGroELs. Trap(D87K) (green) or trap(N265A) (purple) was added at the same time as ATP (zero time) and the recovery of rhodanese activity was measured. Details are described in Materials and methods. (B) Recovered rhodanese activity after 60 min of the SR398-assisted folding in the presence of various concentrations of trap(D87K) (green) or trap(N265A) (purple) added at the same time as ATP. Materials and methods are the same as in (A). (C) FRET monitoring of the natural escape of rhodanese from the cage. FRET between donor-labelled rhodaneseAlexa and acceptor-labelled trap(D87K)TexasRed (0.1 μM, green; 0.2 μM, orange) or trap(N265A)TexasRed (0.1 μM, purple) were plotted as percent of maximum FRET efficiency. Experimental details are described in Materials and methods. (D) Recovery of rhodanese activity by in-cage folding and out-of-cage folding of the SR398-assisted folding. Curve A, SR398-assisted folding (all activity, black); curve D, in-cage folding (activity recovered in the presence of trap(D87K), green); curve I, in-cage folding ((curve A)–(curve O), red); curve S, spontaneous folding (blue); curve O, out-of-cage folding (activity of the naturally escaped rhodanese that folded in the bulk medium in the absence of trap(D87K), cyan); curve L, leaked native rhodanese (activity in the bulk medium in the presence of trap(D87K), grey).
Figure 3
Figure 3
GroEL residues that can interact with rhodanese in the cage. (A) Solvent-accessible surface of the GroES-associated GroEL ring in the GroEL–GroES–(ADP)7 complex (PDB code 1AON) viewed from outside, inside, close-up to the GroEL/GroES-binding site from inside and above. GroEL and GroES subunits are coloured in green and orange, respectively. The residues mutated to cysteine are labelled and coloured (hydrophilic, cyan; acidic, red; basic, blue; hydrophobic, yellow). The putative polypeptide-binding site is depicted by a red circle. (B) Cross-linking between rhodaneseHis6 (Rho) and cysteine-introduced SR398 mutants. Diamide was added to form disulphide bonds before (ATP−) or at 0.1 min after the addition of GroES and ATP (ATP+). Non-reducing SDS–PAGE gels were stained with CBB. (C, D) Time course of the cross-linking between rhodaneseHis6 and SR398(R231C) (C) or SR398(V264C) (D) during the assisted folding. After the folding reaction was started, aliquots were oxidized by diamide at indicated times. Time courses of cross-linked products (x-link, closed squares) and free rhodaneseHis6 (free, open squares), and rhodanese activity (closed circles) are plotted.
Figure 4
Figure 4
Increase and decrease of natural escape from SR398 variants. (A) Natural escape of fluorescently labelled rhodaneseAlexa during the assisted folding by SR398(Y203C), NEM-labelled SR398 (SR398(Y203C)NEM), SR398(F44C) and pyrene-labelled SR398 (SR398(F44C)pyrene). Fluorescence of Alexa was monitored as in Figure 1D. (B–E) Time course of folding of rhodanese assisted by SR398(Y203C) (B), SR398(Y203C)NEM (C), SR398(F44C) (D) and SR398(F44C)pyrene (E). Colour patterns of curves were the same as in Figure 2D. Curve A, assisted folding; curve I, in-cage folding ((curve A)–(curve O)); curve O, out-of-cage folding; curve L, leaked native rhodanese. (F, G) Expression and activity of rhodanese in E. coli cells expressing wild-type GroEL, SR398(F44C) or SR398(Y203C). A plasmid containing the groE gene (pTrc-GroE) and another inserted with or without the rhodanese gene (pACYC) were introduced into an E. coli strain MGM100, whose expression of the chromosomal groE gene was dependent on arabinose. The gene contained in each plasmid is shown at the bottom of (G). The lane numbers in (F) are the same as those in (G). Cells were cultured to OD600∼1.5 in the presence of IPTG and in the absence of arabinose. Harvested cells were disrupted by sonication. Proteins in the lysate of disrupted cells were analysed with CBB-stained SDS–PAGE (F, upper panel). Expression of rhodanese (native and non-native) was assessed by western blotting with anti-rhodanese antibody (F, lower panel) and the band intensity was plotted (G, black bars). Native rhodanese contained in the lysate was assessed by the rhodanese activity (G, grey bars). Values are normalized to those of lane 2 (wild-type GroEL and rhodanese). Three independent determinations were averaged.
Figure 5
Figure 5
Denatured DHFR and Rubisco protrude during the chaperonin-assisted folding. (A, B) Interference with the SR1-assisted folding of DHFR (A) and Rubisco (B) by trap(N265A) (5 μM for DHFR(E161C) and 2 μM for Rubisco) added at the times indicated by arrows. Time course of cross-linking between DHFR(E161C) and Rubisco with SR398(R231C) during the assisted folding is also shown. After the folding reaction was started, aliquots were oxidized by diamide at the indicated times. The band intensity of cross-linked products (DHFR) or non-cross-linked monomers (Rubisco) is plotted. (C, D) Conjugation of DHFR (C) and Rubisco (D) with trap(N265A) during the SR1-assisted folding. The fluorescently labelled DHFRAlexa (C) or RubiscoAlexa (D) was used as a substrate protein for the SR1-assisted folding in the absence (black) or presence of trapGroELs (trap(D87K), green; trap(N265A), purple). After a 30-min incubation, the solutions were applied to a gel-filtration column and the fluorescence of Alexa was monitored. NADPH (0.1 mM) was included in the gel-filtration buffer when DHFRAlexa was analysed.
Figure 6
Figure 6
Interference with the GroEL(D398A)-assisted folding by the antibody and streptavidin. (A, C) Interference with GroEL(D398A)-assisted folding of rhodanese (0.1 μM) by anti-rhodanese antibody (4 μM) (A) or streptavidin (10 μM) (C) added at the time indicated. In (C), we used biotinylated rhodanese, whose assisted folding was slower than non-labelled rhodanese. (B) Conjugation of rhodanese with anti-rhodanese antibody during the GroEL(D398A)-assisted folding. The fluorescent rhodaneseAlexa was used as in Figure 1D. After a 30-min incubation, the solutions were applied to a gel-filtration column and the fluorescence of Alexa was monitored. (D) Transmission electron micrographic images of the GroEL(D398A)–GroES–biotinylated rhodanese complex decorated with streptavidin–gold colloid conjugate (left panel). Upon addition of GroES and ATP to the GroEL(D398A)–biotinylated rhodanese complex, endogenous cysteines in denatured rhodanese were cross-linked each other by diamide to prevent the forced escape. After the streptavidin–gold colloid conjugate was added, the GroEL(D398A) complex was purified with gel filtration and loaded on a carbon grid. After a rinse with water, the proteins were negatively stained by 2% of uranyl acetate and were observed. The 22 images of side views of chaperonins with bound gold particle were classified by the location of gold particle-binding sites (near GroEL/GroES interface region or near equatorial region) and shown as a bar graph (right panel).
Figure 7
Figure 7
Mechanistic model of the chaperonin-assisted folding. One of two rings of GroEL is depicted and the turnover of ATP hydrolysis is not included in this model. Upon GroES binding, the ternary complex (D) is formed in which denatured protein (blue curve) is still interacting with GroEL's apical regions nearby the GroEL/GroES interface, protruding partly outside. The D state is a rate-limiting intermediate of folding in the cage. The life-time of D in the case of assisted folding of rhodanese is ∼8 min. The conformation of the polypeptide in the D state is considered to be flexible. Then, the polypeptide becomes free from interaction with GroEL residues and is released into the cage (in-cage release) or into the outside bulk medium (natural escape). Folding state of the polypeptide just after the in-cage release in the T state is not known, but the polypeptide completes folding very rapidly (T → N). The polypeptide that escapes to the bulk medium is in a denatured state (E), and starts folding in a spontaneous manner.
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