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.

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