ATP-Bound States of Groel Captured by Cryo-Electron Microscopy

Cell, Vol. 107, 869–879, December 28, 2001, Copyright 2001 by Cell Press ATP-Bound States of GroEL Captured by Cryo-Electron Microscopy Neil A. Ranson,1,5 George W. Farr,2,3 characterized of which is the GroEL/GroES system in Alan M. Roseman,1,6 Brent Gowen,4,7 E. coli (Hartl, 1996; Ranson et al., 1998; Sigler et al., Wayne A. Fenton,3 Arthur L. Horwich,2,3 1998). The essential physiological role of chaperonins, and Helen R. Saibil1 together with the experimental accessibility of the GroE 1Department of Crystallography system, has stimulated intensive biochemical and theo- Birkbeck College London retical analysis. The resulting information may provide Malet Street a conceptual model for ring complexes which harness London WC1E 7HX the energy of nucleotide binding for many cellular func- United Kingdom tions. 2 Howard Hughes Medical Institute and The double-ring GroEL binds nonnative proteins in a 3 Department of Genetics central cavity, then mediates productive folding inside Yale School of Medicine a sequestered space formed when ATP and GroES bind New Haven, Connecticut 06510 to the same ring as polypeptide. Each GroEL subunit 4 Biochemistry Department comprises an equatorial, ATP binding domain joined by Imperial College a hinge region to a slender intermediate domain that in Exhibition Road turn has a hinge connection to an apical domain mediat- London SW7 2AY ing polypeptide binding (Braig et al., 1994). The two United Kingdom apposed rings of equatorial domains hold the cylindrical oligomer together through both intra- and inter-ring contacts. The cooperative binding of ATP to a GroEL ring allows Summary rapid binding of GroES to that ring, converting it from a polypeptide-accepting to a folding-active state. At the The chaperonin GroEL drives its protein-folding cycle same time, ATP binding allosterically discharges the li- by cooperatively binding ATP to one of its two rings, gands from the opposite ring. These events and the series priming that ring to become folding-active upon GroES of states in the chaperonin reaction cycle are depicted binding, while simultaneously discharging the pre- in Figure 1. Figures 1a and 1b represent states that vious folding chamber from the opposite ring. The have been extensively studied in vitro to characterize GroEL-ATP structure, determined by cryo-EM and the chaperonin system, but are unlikely to form part atomic structure fitting, shows that the intermediate of the in vivo cycle. A nonnative polypeptide binds to domains rotate downward, switching their intersub- hydrophobic sites on the apical domains of an open unit salt bridge contacts from substrate binding to ATP GroEL ring (Figure 1a or 1e). Positively cooperative bind- binding domains. These observations, together with ing of ATP to one of the two GroEL rings promotes a the effects of ATP binding to a GroEL-GroES-ADP conformational change in the chaperonin (Figure 1b or complex, suggest structural models for the ATP- 1e) and is a prerequisite for binding of the cochaperonin induced reduction in affinity for polypeptide and for GroES to that ring (Chandrasekhar et al., 1986; Gray and cooperativity. The model for cooperativity, based on Fersht, 1991; Jackson et al., 1993; Todd et al., 1994). switching of intersubunit salt bridge interactions Negative cooperativity with respect to ATP binding re- around the GroEL ring, may provide general insight sults in asymmetric complexes, with the two rings into cooperativity in other ring complexes and molecu- adopting different conformations (Yifrach and Horovitz, lar machines. 1995; Burston et al., 1995). Binding of GroES to the ATP- bound ring brings about major conformational changes Introduction that involve rigid body movements of the intermediate and apical domains, the latter elevating 60Њ and twisting A variety of ATP and GTP-dependent molecular ma- 90Њ clockwise. As a result, the hydrophobic polypeptide chines perform many of the essential macromolecular binding sites move into the interfaces between GroEL processes in living cells, including those critically in- subunits, and the walls of the folding chamber become volved in information transfer: transcription, translation, hydrophilic. The binding of ATP and GroES thus acts to and protein folding. Machines mediating the last of these release the substrate polypeptide from its apical binding steps have been the most recently recognized, and their sites into a hydrophilic folding chamber capped by the nucleotide-directed mechanisms pose fascinating prob- dome-shaped GroES (Figure 1c; Roseman et al., 1996; lems (Bukau and Horwich, 1998). Of particular interest Xu et al., 1997). The encapsulated polypeptide is free are the ring assemblies known as chaperonins, the best to undergo productive folding in this protected space (Weissman et al., 1995, 1996; Mayhew et al., 1996; Rye (s 10ف et al., 1997). Subsequently, ATP hydrolysis (t1/2 5 Correspondence: [email protected] weakens the binding of GroES (Figure 1d), and the free 6 Present address: Medical Research Council, Laboratory of Molecu- GroEL ring can now bind ATP and nonnative polypeptide lar Biology, Hills Road, Cambridge CB2 2QH, United Kingdom. 7 Present address: Division of Structural Biology, Wellcome Trust (Figure 1e), providing the signal for release of GroES Centre for Human Genetics, University of Oxford, Roosevelt Drive, and polypeptide and enabling GroES to bind to the ATP- Oxford OX3 7BN, United Kingdom. bound ring, forming a new folding-active complex (Fig- Cell 870 Figure 1. Schematic Diagram of GroEL Func- tional States (a) Nonnative polypeptide substrate (wavy black line) binds to an open GroEL ring. (b) ATP binding to GroEL alters its conformation, weakens the binding of substrate, and per- mits the binding of GroES to the ATP-bound ring. (c) The substrate is released from its binding sites and trapped inside the cavity formed by GroES binding. (d) Following en- capsulation, the substrate folds in the cavity and ATP is hydrolysed. (e) After hydrolysis in the upper, GroES-bound ring, ATP and a second nonnative polypeptide bind to the lower ring, discharging ligands from the upper ring and initiating new GroES binding to the lower ring (f) to form a new folding active complex on the lower ring and complete the cycle. (f) and (c) are related by 180Њ rotation. Thus, the rings of GroEL alternate between folding-active and polypeptide-accepting states. (a) and (b) are shown in brackets to indicate that they represent states that are functional in vitro, even though they are unlikely to be significantly populated in vivo. The in vivo cycle includes cycling between the asymmetric states (c)–(f). ure 1f; Rye et al., 1997, 1999). The asymmetric forms ward rotation of the intermediate domains in the ATP- shown in Figures 1c–1f are likely to represent in vivo bound ring communicates a conformational change states, with Figure 1d as the predominant acceptor state around the ring. The apical domains, released from an for nonnative polypeptide. initial salt bridge constraint, twist counterclockwise by 115Њ clockwise excursion of theف 25Њ, requiring a netف It is clear that ATP binding plays a pivotal role in chaperonin function, but the structural consequences substrate binding sites during the subsequent encapsu- of ATP binding remain poorly understood. Cryo-EM lation of folding protein by GroES binding. Owing to studies of GroEL-nucleotide complexes at 30 A˚ resolu- tilts of the equatorial domains, the inter-ring interface tion all showed extension and twisting of the apical is distorted, suggesting a pathway for propagating neg- domains and asymmetry between the rings (Roseman ative cooperativity to the opposite ring. A similar coun- et al., 1996, 2001; White et al., 1997). Although the crystal terclockwise twist is also observed when ATP binds to structure of GroEL containing the nonhydrolyzable ATP the open ring of GroES-ADP7-GroEL to form GroES- analog ATP␥S has been determined, both rings were ADP7-GroEL-ATP7 (corresponding to Figure 1e), sug- fully occupied with nucleotide (Boisvert et al., 1996). gesting a mechanism by which ATP might trigger disso- Perhaps as a consequence, the structure did not capture ciation of the GroES-capped folding chamber on the the domain rotations or asymmetry between the two opposite ring. GroEL rings that had been observed in solution. Even more problematic, it has been observed that nonhydro- Results lyzable ATP analogs can neither promote productive folding of stringent substrates nor drive dissociation of Solution Structures of Unliganded GroEL folding-active complexes, suggesting that the analogs and GroEL(D398A)-ATP cannot mimic the stereochemical action of ATP on To improve the resolution of our EM maps to 10 A˚ ,it GroEL (Rye et al., 1997). Thus, the question remains as was necessary to collect large datasets on a microscope to what conformational changes are accomplished by with a coherent source and correct for electron optical the action of ATP that enable GroES binding to the ATP- distortions (Bo¨ ttcher et al., 1997; Conway et al., 1997). bound ring and discharge of ligands from the opposite To validate our microscopy and single particle image ring. The available low-resolution cryo-EM images have processing methods, we determined the structure of suggested that the apical domains in a GroEL-ATP com- unliganded GroEL and compared it to the known crystal plex move partway toward the GroES-bound state structure. The resulting GroEL density map is shown in (Roseman et al., 1996), but the details and mechanisms Figure 2a. The isolated domains were docked separately of these movements are unclear. Moreover, the nature as rigid bodies into each ring of the EM density map of the contacts mediating the cooperative action within using the program DockEM (Roseman, 2000), which pro- and between GroEL rings remains obscure. vided an excellent fit. The map resolution was 10.8 A˚ at To further probe the conformational changes pro- a Fourier shell correlation of 0.5 and 7.9 A˚ at the 3␴ moted by ATP binding to GroEL, we have examined cutoff.

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