Requirement for Binding Multiple Atps to Convert a Groel Ring to the Folding-Active State

Requirement for binding multiple ATPs to convert a GroEL ring to the folding-active state

Eli Chapmana, George W. Farrb,c, Wayne A. Fentonc, Steven M. Johnsona, and Arthur L. Horwicha,b,c,1

aThe Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037; and bHoward Hughes Medical Institute and cDepartment of Genetics, Yale School of Medicine, Boyer Center, 295 Congress Avenue, New Haven, CT 06510

Contributed by Arthur L. Horwich, October 22, 2008 (sent for review October 9, 2008)

Production of the folding-active state of a GroEL ring involves reflected in a structure of GroEL–GroES–ADP–AlF3 (11), initial cooperative binding of ATP, recruiting GroES, followed by affords a critical level of energy and cooperativity. large rigid body movements that are associated with ejection of The question remains, however, of how many subunits must bound substrate protein into the encapsulated hydrophilic cham- cooperatively bind ATP to recruit the collective of subunits in a ber where folding commences. Here, we have addressed how ring to the folding-active state. For example, does binding a many of the 7 subunits of a GroEL ring are required to bind ATP to single ATP to a ring bring about such movement and activation drive these events, by using mixed rings with different numbers of of folding? Or, at the other extreme, is there a requirement to wild-type and variant subunits, the latter bearing a substitution in bind ATP to each subunit to produce the collective movement the nucleotide pocket that allows speciﬁc block of ATP binding and that activates folding? In other words, how redundant is the turnover by a pyrazolol pyrimidine inhibitor. We observed that at system with respect to its ATP requirement? Here, we have been least 2 wild-type subunits were required to bind GroES. By con- able to address this question by using a small-molecule chemical trast, the triggering of polypeptide release and folding required a inhibitor that specifically blocks ATP binding and hydrolysis by minimum of 4 wild-type subunits, with the greatest extent of a subunit variant containing an amino acid substitution in the refolding observed when all 7 subunits were wild type. This is GroEL nucleotide pocket. Using mixed rings composed of both consistent with the requirement for a ‘‘power stroke’’ of forceful wild-type and variant subunits, the chemical compound, and apical movement to eject polypeptide into the chamber. ATP, we could assess the number of subunits required for steps of both GroES association and triggering productive folding. ͉ ͉ ͉ ͉ chaperonin chemical inhibitor nucleotide protein folding Results pyrazolol pyrimidine A Pyrazolol Pyrimidine Competitively Blocks ATP Binding and Hydro- lysis by a GroEL Variant Substituted in the Nucleotide Pocket, I493C. n the cell, ATP and GTP are ubiquitously used as molecular To investigate the number of subunits in a GroEL ring that must Itriggers that drive a host of cellular functions. In particular, bind ATP to activate folding, we sought a small molecule ATP drives the final step of information transfer for many genes, inhibitor that could specifically prevent binding of ATP to a the step of chaperonin-mediated protein folding (1, 2). The variant GroEL subunit. This would enable probing mixed rings GroEL double ring machine, for example, is directed into its containing various proportions of variant and wild-type subunits folding-active state by the concerted binding of ATPs to the 7 in the presence of inhibitor to assess the requirements for ATP subunits of a substrate polypeptide-bound ring (3). ATP binding binding in driving GroES association and in initiating productive in the equatorial domains of these subunits drives rigid body folding. The strategy used for isolating an orthogonal inhibitor– movements of the apical domains, which bear the hydrophobic variant GroEL pair corresponded to that implemented by polypeptide-binding surface lining the opening to a ring (4, 5). Shokat and coworkers in studying actions of specific protein

In particular, small ATP-directed elevation and twisting move- kinases (21): side chains at the position of the base in the GroEL BIOCHEMISTRY ments serve to recruit the cochaperonin lid structure, GroES (6). nucleotide pocket were shortened to alanine or cysteine, and a This triggers further large movements of the apical domains, variety of pyrazolol pyrimidine derivatives were then tested for Ϸ60° elevation and Ϸ120° clockwise twist, that release the the ability to block ATP hydrolysis in vitro by the purified substrate protein off the dislocating hydrophobic surface, effec- homooligomeric GroEL variants (E.C., G.W.F., K. Furtak, and tively ejecting the substrate into a hydrophilic GroES- A.L.H., unpublished work). Strong inhibition of hydrolysis was encapsulated chamber where productive folding ensues (4, 5, obtained with a variant, I493C, and a cyclopentane-carboxamide 7–14). This step of ATP-mediated activation requires less than derivative, EC3016 [Fig. 1 A and B; see supporting information 1 s (9, 12). Productive folding then proceeds for Ϸ10 s followed (SI) Methods and Fig. S1 for synthesis). by ATP hydrolysis and subsequent ATP binding to the opposite I493C behaved as a bona fide variant of GroEL. In vivo, ring, which leads to rapid dissociation of GroES and substrate expression of the variant from a trc promoter-driven GroESL protein (Ͻ1 s) (9, 15). operon carried on a pBR322 plasmid in the absence of induction ATP is specifically required for the activation of folding (16, led to a level of I493C GroEL corresponding to the normal level 17), with ADP unable to support release of polypeptide into the of chromosomally expressed wild-type GroEL, and this pro- folding chamber despite the ability to produce the movements duced rescue of GroEL-deficient LG6 cells (22). In addition, the that enable stable binding of GroES (7, 9, 11, 18). In particular,

the apical domains of GroEL are observed to move more slowly Author contributions: E.C. and A.L.H. designed research; E.C., G.W.F., W.A.F., S.M.J., and in the presence of bound substrate protein during the formation A.L.H. performed research; E.C. contributed new reagents/analytic tools; E.C., W.A.F., of ADP–GroES complexes, failing to release the substrate, S.M.J., and A.L.H. analyzed data; and E.C. and A.L.H. wrote the paper. reflecting that ADP-driven movements may not be as forceful The authors declare no conﬂict of interest. (18). In addition, ATP binds cooperatively to GroEL whereas Freely available online through the PNAS open access option. ADP does not (19). GroEL mutations that abolish this cooper- 1To whom correspondence should be addressed. E-mail: [email protected]. ative action of ATP reduce the rate of productive folding (20). This article contains supporting information online at www.pnas.org/cgi/content/full/ Thus, the presence of the ␥-phosphate, conferring a host of 0810657105/DCSupplemental. hydrogen bonds with the surrounding nucleotide pocket, as © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810657105 PNAS ͉ December 9, 2008 ͉ vol. 105 ͉ no. 49 ͉ 19205–19210 Downloaded by guest on September 29, 2021 1B) because it allowed easy detection of this subunit in coex- pression studies by its faster migration than I493C in SDS/ PAGE. 532⌬ exhibits a slightly reduced rate of steady-state ATP turnover relative to nondeleted GroEL, as expected based on recent observations that steady-state ATP turnover scales approximately with C terminus tail length of GroEL (23). If anything, EC3016 slightly stimulated the rate of steady-state turnover of both wild- type and 532⌬. This may be a function of nonspecific association of the inhibitor with the hydrophobic polypeptide-binding surface of the GroEL apical domains, leading to allosteric activation of ATP turnover. Consistent with such behavior, we observed that virtually all of the other pyrazolol pyrimidine compounds produced the same mild stimulation of GroEL ATPase activity. To address further the nature of inhibition by EC3016, initial rates of ATP hydrolysis were measured with varying concentrations of ATP and EC3016, plotted as a set of curves in Fig. 1C. For each curve, the pattern of increasing rate of turnover with respect to ATP concentration followed by a decrease fits the nested-cooperativity model originally proposed by Yifrach and Horovitz (3), entailing cooperative binding of ATP within a GroEL ring and negative cooperativity between rings. Thus, cooperative ATP binding is maintained by I493C subunits. With increasing concentration of EC3016 compound, the curves were progressively displaced downward. Extensive modeling of the data supported a mechanism of competitive inhibition, but other models cannot be completely excluded. Consistent, however, with the notion that inhibition occurs at the level of competition for the nucleotide pocket, steady-state turnover by a single ring version of I493C was also strongly inhibited by EC3016 (Fig. 1D).

Mixed-Ring Assemblies Containing Various Proportions of Wild-Type and I493C Subunit. To analyze requirements for ATP binding, we programmed expression of mixed-ring GroEL complexes containing various proportions of 532⌬ subunits and I493C subunits from separate plasmids with either ara or trc promoters (Fig. 2). This was accomplished by varying the relative levels of induction with different concentrations of arabinose and IPTG in the medium (Fig. 2). Relative induction levels were adjusted based on analyses in SDS/PAGE, where 532⌬ migrated faster than I493C. The complexes from cultures with various relative levels of induction were then purified chromatographically and, to determine more precisely the relative content of the 2 different subunits, mass spectrometry (MS) was carried out on the purified complexes employing ‘‘standards’’ produced by mixing different relative known amounts of purified homooligomers of 532⌬ and I493C (see, for example, Fig. S2). The relative induc- tions and amounts of the 2 subunits, designated W for 532⌬ (wild type-like) and M for I493C (mutant), are shown in Fig. 2. The mixed complexes are referred to based on the average relative numbers per ring of W and M subunits. For those complexes in which either 1 or 2 mutant subunits Fig. 1. A pyrazolol pyrimidine inhibits ATP hydrolysis by a variant of GroEL, were desired per ring (W6M1 and W5M2), a further strategy was I493C, containing an amino acid substitution in the nucleotide pocket. (A) used. This involved production of rings as covalent assemblies Cyclopentane carboxamide derivative, EC3016. Synthesis of this compound is from 7-fold tandemized coding sequences (24). In the case of described in SI Methods and diagrammed in Fig. S1.(B) Steady-state ATP W6M1, 6 wild-type coding sequences were followed by an I493C turnover by 3 chaperonin molecules, wild-type GroEL, a C-terminally trun- coding sequence (Fig. 2). Likewise, 2 different covalent W5M2 ⌬ cated variant 532 , and I493C, in the absence and presence of EC3016. (C) complexes were produced, 1, U1U7 493C, as a consecutive Initial rates of ATP hydrolysis by I493C, measured as a function of concentration of ATP and of EC3016. (D) Steady-state ATP turnover by single-ring arrangement of variant subunits within the ring, and another, derivatives in the absence and presence of EC3016. U1U4 493C, as an arrangement of variant subunits across the ring from each other. In all 3 cases, the covalent connections of the assembly were gently proteolytically clipped after purification to purified I493C chaperonin exhibited normal cooperative ATP relieve steric strain (see Experimental Procedures and ref.24). hydrolysis behavior in vitro in the absence of inhibitor, resem- bling that of wild-type GroEL (Fig. 1C). In the presence of 50 In the Presence of EC3016, Mixed Complexes W1M6, W2M5, and W3M4 ␮M EC3016, however, ATP turnover was blocked. By contrast Fail to Drive ATP–GroES-Mediated Folding, W4M3 and W5M2 Exhibit with its strong effects on I493C chaperonin, EC3016 exhibited no Modest Refolding Activity, and W6M1 Exhibits Substantial Refolding. detectable inhibition of either wild-type GroEL or a C- The various mixed complexes were next studied for the ability to terminally deleted version, 532⌬, used here as ‘‘wild-type’’ (Fig. mediate refolding of the stringent, ATP–GroES-dependent

19206 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810657105 Chapman et al. Downloaded by guest on September 29, 2021 Fig. 2. Mixed-ring assemblies used to delineate ATP requirements. Stochas- tically produced ring assemblies that contain different numbers of wild-type 532⌬ GroEL subunit, designated W with a number (green), and I493C compound-inhibitable subunit, designated M with a number (red), were produced by using 2 different regulatable plasmids, 1 an ara-regulated plasmid driving 532⌬ (green) and a 2nd a trc-regulated plasmid driving I493C (red). The double

transformants were supplied with varying levels of the 2 inducers arabinose BIOCHEMISTRY (Wt) and IPTG (I493C). In the case of W7 and M7, transformants containing only the individual plasmids were induced. In the case of the other plasmids, the relative levels of subunit in a ring were determined by MS of the purified complexes, compared with standards mixing together known amounts of the Fig. 3. Rhodanese refolding by mixed-ring complexes, carried out in the pure tetradecamers of M7 or W7. Covalent ring assemblies with fixed subunit absence (red) and presence (blue) of EC3016. Binary complexes were formed composition and arrangement, W5M2 and W6M1, were produced from 7-fold between the indicated chaperonins and rhodanese diluted from guanidine tandemized GroEL wild-type and I493C coding sequences as shown, and the HCl, and then GroES and ATP were added. The recovery of rhodanese enzyme double rings were purified and the covalent linkages proteolytically clipped activity was measured at various times thereafter. Each complex was tested (see Experimental Procedures). multiple times; representative experiments are shown.

substrate proteins, rhodanese (Fig. 3) and malate dehydrogenase 20%. This lesser extent of recovery than W4M3 for the 2 covalent ␮ (MDH) (Fig. S3), in the absence and presence of 50 M EC3016. complexes, even though they contained an additional wild-type For these studies, binary complexes were formed between subunit, suggests that the stochastically produced complexes denatured substrate protein diluted from guanidine HCl and the comprise a range of subunit composition that leads to an various mixed-ring assemblies, then ATP and GroES were overestimate of the extent of folding function relative to the added. As shown in the panels in Fig. 3, in the absence of defined composition of the covalent assemblies. Concerning the inhibitor all of the mixed complexes were proficient in mediating 2 different W5M2 complexes, it appears that the arrangement of refolding of rhodanese upon addition of ATP–GroES. In the variant subunits within a ring does not have major effect on the presence of EC3016, however, no refolding was mediated by extent of refolding activity. Finally, the covalent W6M1 complex I493C homooligomer (M7), or by W1M6, W2M5, and W3M4 mediated substantial refolding in the presence of EC3016 (H and complexes (B–E). Only a modest amount of recovery was Fig. S3). For rhodanese and MDH, both the rate and extent of supported by W4M3 (F). Likewise, both covalent W5M2 com- recovery were Ϸ40% that of the wild-type complex. Overall, plexes produced modest recovery of native rhodanese in the these results indicate that binding of ATP to fewer than all 7 presence of ATP/GroES and EC3016 (G), amounting to 15– subunits can be sufficient to drive productive folding of stringent

Chapman et al. PNAS ͉ December 9, 2008 ͉ vol. 105 ͉ no. 49 ͉ 19207 Downloaded by guest on September 29, 2021 substrates like rhodanese and MDH, albeit less efficiently. Considering the activities of the various assemblies, both stochastically and covalently produced, it seems that binding of ATP to at least 4 or 5 subunits is required for driving any significant degree of productive folding and that refolding rate and yield are progressively increased as ATP occupancy is increased, with full occupancy of all 7 subunits producing optimal refolding.

GroES-Binding Requirement: Two or More ATP-Binding-Proficient Sub- units Are Sufficient. To address the nature of defective folding by M7, W1M6, W2M5, and W3M4 complexes, we first asked whether these complexes could bind the cochaperonin GroES to form a cis-folding chamber. This was measured by incubation of the respective complexes in the presence of ADP with a fluorescent GroES, bearing fluorescein on a cysteine added to the externally facing C terminus of the GroES subunit (GroES98C). The products of the incubation were then analyzed by gel filtration with in-line fluorescence detection. In the absence of EC3016, all of the complexes could efficiently bind GroES (see Fig. S4). In the presence of EC3016, binding of GroES by wild type was unaffected whereas both I493C (M7) homooligomer and W1M6 bound only a tiny fraction of the input GroES (Fig. S4). When 2 wild-type subunits were present (W2M5), Ϸ50% of the input GroES was now bound, and when 3 or more subunits were present the binding of GroES became as efficient as with wild type (Fig. S4). Making the assumption that EC3016 com- petes with ADP in the same manner as with ATP, it appears that recruitment of adenine nucleotide to 2 or more subunits is sufficient to enable stable binding of GroES to a GroEL ring. Fig. 4. Mixed-ring complex W3M4, able to bind GroES but unable to refold rhodanese, fails to release bound rhodanese into the central cavity upon Basis to Defective Folding by W2M5 and W3M4 Despite Ability to Bind addition of ATP/GroES in the presence of EC3016. (A) Models for behavior of GroES: No Release of Rhodanese from the Cavity Wall of W3M4 W3M4 upon binding ATP–GroES (Center) and upon subsequent binding of Complex Upon Incubation with ATP–GroES and EC3016. Although ATP in trans (Right), if it were able to release the bound substrate into the cis GroES could bind in the presence of inhibitor to mixed com- cavity (Upper) or if it were unable to release the substrate off of the cavity wall plexes with 2 or 3 wild-type subunits, little or no productive (Lower). In the latter case, if the substrate fails to be ejected into the cis-cavity, folding ensued. What is the basis for such formation of a folding it remains associated at the step of trans-triggered release and fails to transfer chamber but failure to trigger productive folding? We surmised to the trap molecule. (B) Measurement of transfer of [35S]rhodanese from that whereas GroES could bind to these complexes, the substrate wild-type or W3M4 complex to a 337/349 trap mutant of GroEL, separable by polypeptide had failed to be ejected off of the cavity wall into the anion exchange chromatography, after addition of ATP–GroES in the absence or presence of EC3016. The reaction was allowed to proceed for 5 min, folding chamber. Such behavior resembles that observed when, amounting to multiple reaction cycles, and the mixture was then chromato- for example, GroEL–rhodanese binary complexes are incubated graphed and the respective chaperonin fractions subjected to scintillation with ADP and GroES, forming a substrate–GroEL–GroES counting. From wild-type complex Ϸ45% of the input rhodanese transferred ternary complex in which rhodanese remains bound to the to trap regardless of the presence of EC3016, whereas no transfer above GroEL cavity wall (7, 18). To assess here whether polypeptide background (Ϸ20%) was observed from W3M4 when EC3016 was present. A remained associated with the cavity wall of the mixed-ring representative experiment is shown. complex, we asked whether it could transfer to an added GroEL ‘‘trap’’ molecule upon ATP–GroES binding (Fig. 4A). In earlier studies of wild-type GroEL–GroES folding reactions, this was transferred from wild-type GroEL to trap over a period of 5 min observed to occur rapidly, indicating that the polypeptide was both in the absence and presence of EC3016, consistent with released into the cis-cavity, attempted folding, but then was released earlier studies (Fig. 4B). A similar amount of rhodanese was also into solution upon dissociation of the cis-complex as a nonnative transferred from the W3M4 complex to trap in the absence of Ϸ molecule that could be captured by trap (ref. 25; notably, only 5% EC3016, corresponding to fully productive folding of rhodanese of rhodanese molecules reached the native state per folding cycle, Ϸ by this complex in the absence of inhibitor. In the presence of with 95% released in nonnative form). EC3016, however, W3M4 failed to transfer any rhodanese above We used 35 S-radiolabeled rhodanese as substrate, first forming a background amount (15–20%) that occurs during anion ex- binary complexes with either wild-type or W3M4 complex. The change chromatography (Fig. 4B and ref. 25). This failure to GroEL mutant G337S/I349E, which binds nonnative rhodanese but fails to release it and is separable from GroEL (and also from release rhodanese was not caused by a simple failure of ATP W3M4) in anion exchange chromatography, was added as a trap, binding in trans in the presence of EC3016 to release GroES thus enabling direct observation upon chromatography of from the cis ring: in a parallel experiment, fluorescent GroES whether rhodanese remained with the W3M4 complex upon was observed by gel filtration with in-line fluorescence to ATP–GroES addition or was transferred to the 337/349 trap. transfer under these conditions to added SR1, functioning as a ATP and GroES were added, and the reaction was allowed to GroES trap (data not shown). We conclude that, in the presence proceed for 5 min before quenching with EDTA. The mixture of EC3016, although GroES can bind to the W3M4 complex, was subjected to anion exchange chromatography and the 35S there is no release of the substrate protein from the cavity wall radioactivity monitored at the elution positions of W3M4 and into the folding chamber, and thus no productive folding can 337/349. We observed that Ϸ45% of the input molecules were ensue.

19208 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0810657105 Chapman et al. Downloaded by guest on September 29, 2021 as substrate polypeptide, which acts as a retarding ‘‘load’’ on apical domain movement, is not present (18). Thus, ADP appears in this context to be an informative nucleotide concerning GroES binding. The advantage to using ADP is that it produces a stable physical association of GroES with GroEL, as opposed to the dynamic association in ATP (26). This allows monitoring complex formation, as here, by gel filtration chromatography. In addition, the effects of EC3016 on nucleotide binding by I493C variant subunits are expected to be the same with respect to ADP as observed with ATP in hydrolysis assays, presumably mediated by binding at the position in the nucleotide pocket normally occupied by the adenine base. The results presented here with the various mixed-ring complexes and EC3016 indicate that the binding of ADP to a single GroEL subunit is not sufficient to recruit GroES, that 2 ADPs produce fractional binding, and that full binding is achieved with 3 or more ADPs bound. The ADP requirement could come from either of 2 different types of behavior. In one model, apical domain mobilization would need to occur for only the few subunits of the ring that are binding ADP, with these apical domains presenting in the freed state, exhibiting the requisite elevation and counterclockwise twist needed to produce an initial GroES-docking event. Then, the binding energy of GroES Fig. 5. Summary of ATP requirements for triggering productive folding: 2 association with the docking apical domains could promote the levels of ATP requirement. At a first level, binding of a single ATP to a GroEL full range of movement of all of the apical domains of the ring. ring is insufficient to trigger stable GroES binding, potentially because of In a second model, there would be a requirement to mobilize all failure to produce the small movements of elevation and twist of the collective of the apical domains of a ring simultaneously, with each of apical domains needed to recruit GroES (see Discussion). Binding of at least undergoing the small elevation and counterclockwise twist to 2 ATPs per ring was required for GroES association as measured here by gel dock GroES. This latter model of all-or-none behavior would filtration (Fig. S4). At a second level, binding of at least 4–7 ATPs per ring was mirror the all-or-none breaking of salt bridges between neigh- needed to produce folding, reflecting the likely need for forceful apical boring subunits required for apical domain movement (6). movement to eject polypeptide off of the elevating and twisting apical domains into the cis-cavity to enable folding to commence. As shown in Fig. Normal mode and modeling studies of Ma and Karplus (27) have 4, in the absence of this power stroke, as in the case of W3M4, release into the indicated this as an operative mechanism because steric clash cavity and productive folding do not occur. would result if, for example, 1 mobilized subunit commenced such movements in the absence of movements of its neighbors. Thus, the simultaneous breaking of all intersubunit bonds seems Discussion to be mandated, and the energy for doing this may only be able The observations presented here concerning the effects of to be derived here as the result of binding ADP to 2 or more restricting ATP binding to varying numbers of the 7 subunits per subunits. GroEL ring allow distinction of different requirements for ATP In energetic terms, our previous calorimetric measurements of binding to drive 2 major actions of ATP (Fig. 5). Binding of ATP ADP binding to a single ring of GroEL (SR1) indicate a release to 2 or more subunits is sufficient to drive GroES cochaperonin of free energy amounting to 6 kcal/mol of ADP bound per mol of rings (11), suggesting that perhaps 12–18 kcal of free energy

binding; but binding to 4 or more subunits is needed for any BIOCHEMISTRY degree of productive folding, in particular for driving ejection of (binding to 2–3 subunits) per mol of rings is sufficient to drive bound substrate protein off of the GroEL cavity wall into the the requisite intersubunit bond breakage and elevation and twist of all of the apical domains of a ring required for GroES binding. GroES-encapsulated folding chamber. ATP Binding to 4 or More Subunits Is Required to Drive Productive Binding of ADP to 2 or More Subunits of a Ring Is Required for GroES Folding: Likely Requirement for a Concerted Apical Power Stroke to Binding, a Likely Requirement for Concordant Movement and Estimate Eject Bound Polypeptide into the Folding Chamber. The observations of Energy Cost. It has been understood that initial ATP (or ADP) that all-variant complex and W1M6 mixed-ring complex were binding to the equatorial binding sites of a GroEL ring is unable to refold rhodanese or MDH substrate proteins in the required to enable the association of GroES cochaperonin presence of ATP and EC3016 are consistent with a requirement through the apical domains of the nucleotide-bound ring (1, 2). by these substrate proteins for encapsulation to fold productively Here, we have been able to deduce that at least 2 subunits of a and with the inability of such complexes to bind GroES effi- ring must bind ADP, and by extension ATP (see below), to ciently, as discussed above. However, the failure of W3M4 and recruit GroES. How does this translate in structural terms? W4M3 to fold these substrates came as something of a surprise. In the setting of wild-type GroEL, cooperative ATP binding These complexes could clearly bind GroES in the presence of to the 7 equatorial domains of an open ring produces small rigid ADP and EC3016 (Fig. S4), but yet no or little productive folding body movements that, among other effects, expose a docking was observed in the presence of ATP and inhibitor (Fig. 3). We surface on the apical domains, as yet unidentified, for recruit- surmised that these complexes were unable to trigger ejection of ment of GroES (6). Initial docking of GroES is then followed by these polypeptide substrates into the cis-folding chamber when large rigid body apical domain movements, 60° elevation and inhibitor was present, and this was borne out by the transfer 120° clockwise twist, which produce the stable domed GroES- experiment in Fig. 4. This is consistent with the requirement for encapsulated end state that has been observed by both cryo-EM an ATP-directed ‘‘power stroke’’ as a forceful apical movement and X-ray studies (4, 5, 9). Notably, this conformational pro- that ejects polypeptide into the cavity (Fig. 5). It would appear gression and end state, produced in Ͻ1 s in ATP, can be almost that we are measuring the number of subunits required to bind as rapidly driven in the nonphysiologic nucleotide, ADP, as long ATP cooperatively to make such movement sufficiently forceful

Chapman et al. PNAS ͉ December 9, 2008 ͉ vol. 105 ͉ no. 49 ͉ 19209 Downloaded by guest on September 29, 2021 to release the substrate. Not until 4 or more ATPs can be bound plexes were purified by standard procedures. The proportion of the subunits is there sufficient energy to drive the substrate off of the wall. in each mixed complex was preliminarily characterized by SDS/PAGE with This forceful release presumably occurs in association with Coomassie blue staining, where the 2 subunits migrate differently, and es- GroES binding because bound polypeptides such as rhodanese tablished by MS with mixtures of known amounts of parent homooligomers as standards (see Fig. S2; see also SI Methods for comments on subunit mixing and MDH are not efficiently ejected into free solution by in vivo and composition of mixed complexes). The covalent W6M1 mixed addition of ATP alone. Here, binding a minimum of 4 ATPs complex was produced by replacing the C-terminal subunit (U7)-coding se- would release at least 60 kcal/mol of rings of free energy based quence in covalent wild-type GroEL (24) with an I493C-containing subunit by on our earlier measurements, including a small contribution (Ϸ5 exchanging a restriction fragment, and the presence of the substitution was kcal/mol) from GroES binding. This energy is supporting the verified by DNA sequencing U7 by antisense priming from downstream vector large apical domain movements needed to eject polypeptide fully sequence. Covalent W5M2 complexes were produced by replacing unit 1 or from its binding sites. These movements must necessarily also be unit 4 in the U7 493C covalent complex with an I493C subunit where the coordinated, as indicated by the studies of Ma and Karplus (27). substitution was marked by an MfeI restriction site adjacent to the substitu- This clearly implies that the force generated by those subunits tion that preserves the coding sequence, allowing identification of desired clones by restriction analysis. Covalent (double-ring) assemblies were ex- occupied with ATP is being coopted to move subunits that have pressed and purified, and the connections between subunits were cleaved not bound nucleotide. In sum, we have observed that the with proteinase K as before (24). Such protease treatment has been shown not cooperative binding of at least 4 ATPs is required to produce the to result in the exchange or rearrangement of subunits in mixed-ring com- energy necessary for any productive release of substrate protein plexes (24). GroEL337/349 and [35S]rhodanese were produced as described in to occur, with increased recovery occurring with greater oc- ref. 25. cupancy and optimal recovery occurring with all 7 subunits GroES binding, ATPase assay, and rhodanese transfer assay were carried out occupied. as described and are detailed in SI Methods. Experimental Procedures ACKNOWLEDGMENTS. We are grateful to Kevan Shokat (University of Cali- ⌬ fornia, San Francisco) for helpful discussion and for supplying pyrazolol pyrimi- Proteins. Coding sequences for I493C and 532 were produced by oligonu- dine compounds for initial testing, to Chi-Huey Wong for supporting the cleotide-directed mutagenesis in pTrc and pBAD-derived vectors, respectively, chemical syntheses, to Krystyna Furtak for technical assistance, and to the and confirmed by DNA sequencing. After growth of TOP10 transformants and National Institutes of Health and Howard Hughes Medical Institute for sup- induction with varying amounts of IPTG and arabinose, the mixed-ring com- porting this work.

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