Structure and Dynamics of the GroEL-binding GroES Mobile Loop

Last modified 12/1/97.

Chaperonin Molecular Structure

Escherichia coli GroEL and GroES are the prototypical chaperonin and co-chaperonin. Chaperonins are ring-shaped molecular chaperone proteins that are found in all organisms and are essential for protein folding and protection from stress. Both the chaperonin and co-chaperonin are 7-fold symmetric oligomers. GroEL is composed of fourteen identical 60 kDa subunits arranged in two stacked rings, and GroES is composed of a seven identical 10 kDa subunits.

top view of GroEL
GroEL. "Top view" C-alpha trace colored by crystrallographic B-factors. Red indicates high mobility; blue low mobility. The inside of the apical domains, where unfolded substrates are thought to bind, are the most mobile.

top view of GroES

GroES. "Top view" C-alpha trace colored by crystallographic B-factors. Only one of seven mobile loops is visible at left. For a closer look examine the kinemage estour_2.kin. [If necessary, first get the kinemage viewer Mage 4.2 ]

side view of GroES and GroEL

GroES and GroEL. "Side view" showing the four-banded appearance of the GroEL "double donut" and dome-shaped GroES. Each GroEL donut is composed of a ring of seven subunits. Each subunit is composed of three domains: apical, intermediate, and equatorial. The equatorial domains are well ordered in the crystal, as indicated by low B-factors (blue). ATP binds to the equatorial domains triggering a conformational change in the intermediate and apical domains that leads to discharge of the folding protein. In addition, allosteric interactions are transmitted from one ring to the other. For example, when GroES binds to one end, binding of GroES to the other end becomes much less favorable. When GroES binds, its seven mobile loops contact the apical domains of GroEL. The one visible mobile loop is probably not in the GroEL-binding conformation, but rather must swing down to make contact with GroEL. The roof of the GroES dome is not very stable, and it is possible that some chaperonin substrates leave the GroEL/GroES right through a hole in the roof!

Mechanism of Protein Folding Assistance

Unfolded proteins bind to hydrophobic surfaces in the central cavities of either GroEL ring. ATP and GroES bind to GroEL forming a cap over the protein-containing cavity and simultaneously causing a conformational change in GroEL that sequesters the hydrophobic surfaces. This releases the protein into the cavity where it is allowed to fold into its native structure as dictated by the primary amino acid sequence. Discharge of the protein into the bulk solvent may occur only when ATP and GroES bind to the opposite ring of GroEL, triggering an unfavorable ring-ring interaction that leads to dissociation of the first GroES and release of the folded protein.

The Mobile Loop

Binding of GroES is mediated by flexible loops 16 amino acids in length that extend from the bottom of the dome-shaped GroES 7-mer. The loops are sufficiently dynamic to observe by solution-phase proton NMR despite being attached to the 70 kDa GroES oligomer. Normally, one cannot resolve proton signals from proteins larger than 20-30 kDa because they tumble too slowly in solution to give reasonably narrow resonance line widths, but in this case the mobile loops wiggle as if only loosely tethered to the slowly tumbling GroES. The mobile loops are immobilized upon formation of the GroEL/GroES/ATP complex. The structure of the GroEL-bound mobile loop was studied using a 20 amino acid synthetic peptide that mimics the mobile loop. Like the mobile loop, this peptide lacks any structure by itself but is induced to form a characteristic beta-hairpin structure upon binding to GroEL. The bound conformation of the peptide cannot be directly observed by NMR in the bound state because GroEL is much larger than the 20-30 kDa ceiling on NMR-approachable molecules. Thus, the structure was determined by analysis of transferred nuclear Overhauser effects (trNOEs) in NMR spectra of the peptide in the presence of GroEL. This technique relies on a rapid equilibrium of the peptide between bound and free states in order that the pattern of magnetization developed by NOEs in the bound state is transferred to the free state where it can be observed. The trNOE approach to studying the structure of peptides bound to large proteins is potentially useful in the design of new drugs that inhibit protein-protein interactions. In our example, we have learned that a cluster of hydrophobic residues are displayed along one side of the GroEL-bound hairpin structure, and we suspect that key glycine residues in the loop peptide are necessary to form the hairpin. These hypotheses are being tested with mutant GroES proteins prepared by recombinant DNA.

Coordinates for the trNOE NMR structure of the central nine residues of the GroES mobile loop are available from the Brookhaven Protein Database under entry 1EGS. This file contains twenty structures generated by molecular dynamics and simulated annealing. Structure #17 is shown below and in Landry et al., 1996. Recently, Xu et al. (1997) solved the X-ray crystal structure of the GroES-GroEL-ADP complex and found the loop in a beta hairpin conformation as we described.

hairpin structure

Alternative Conformations of the Loop and the Entropic Penalty for Binding to GroEL.

The crystal structure of GroES was solved by our collaborators John Hunt, Art Weaver and Hans Deisenhofer at UT Southwestern in Dallas. Six of seven mobile loops were disordered and thus were not observed, but the seventh loop was trapped between GroES molecules in the crystal lattice. Thus we can compare the structure of this loop to the structure of the GroEL-bound loop peptide. Interestingly, they are quite different. Although they both are in hairpin conformations, the GroEL-bound loop turns to the left instead of to the right. These two conformations provide snapshots of the ensemble of conformational states sampled by the GroES mobile loop when not bound to GroEL. Dynamic fluctuation among many conformations is a form of entropy, an energy of randomness. When GroES binds to GroEL, this energy must be given up as the loops adopt the hairpin structure. The result is weaker binding of GroES to GroEL than would have occurred if the loops were rigid. Since GroES and GroEL must bind and dissociate with each cycle of protein folding assistance, we suspect that the GroEL-binding loops have evolved mobility in order to facilitate dissociation of GroES.


This material is based upon work supported by the National Science Foundation under Grant No. MCB-9512711.

Links to Other Chaperonin-Related Sites

The Chaperonin Home Page

The Chaperonin ATPase Cycle: Mechanism of Allosteric Switching, a movie published on the Web by the Saibil group and associated with the article by Alan M. Roseman, Shaoxia Chen, Helen White, Kerstin Braig, and Helen R. Saibil in the October 18, 1996 issue of Cell.


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Landry, S. J., and Gierasch, L. M. (1994). Polypeptide Interactions with Molecular Chaperones and the Relationship to In Vivo Protein Folding. Ann. Rev. Biophys. Biomolec. Struct. 23, 645-669.

Todd, M. J., Viitanen, P. V., and Lorimer, G. H. (1994). Dynamics of the chaperonin ATPase cycle: Implications for facilitated protein folding. Science 265, 659-666.

Weissman, J. S., Kashi, Y., Fenton, W. A., and Horwich, A. L. (1994). GroEL-mediated protein folding proceeds by multiple rounds of binding and release of nonnative forms. Cell 78, 693-702.

Braig, K., Otwinowski, Z., Hegde, R., Boisvert, D. C., Joachimiak, A., Horwich, A. L., and Sigler, P. B. (1994). The crystal structure of the bacterial chaperonin GroEL at 2.8 angstrom. Nature 371, 578-586.

Fenton, W. A., Kashi, Y., Furtak, K., and Horwich, A. L. (1994). Residues in chaperonin GroEL required for polypeptide binding and release. Nature 371, 614-619.

Weissman, J. S., Hohl, C. M., Kovalenko, O., Kashi, Y., Chen, S. X., Braig, K., Saibil, H. R., Fenton, W. A., and Horwich, A. L. (1995). Mechanism of GroEL action: Productive release of polypeptide from a sequestered position under GroES. Cell 83, 577-587.

Hunt, J. F., Weaver, A. J., Landry, S. J., Gierasch, L., and Deisenhofer, J. (1996). The crystal structure of the GroES co-chaperonin at 2.8 angstrom resolution. Nature 379, 37-45.

Landry, S. J., Taher, A., Georgopoulos, C., and van der Vies, S. M. (1996). Interplay of structure and disorder in co-chaperonin mobile loops. Proc. Natl. Acad. Sci. U. S. A. 93, 11622-11627.

Saibil, H. R. (1996). Chaperonin structure and conformational changes. In Cell Biology, A Series of, Monographs: Chaperonins, R. J. Ellis, ed. (San Diego: Academic Press Inc), pp. 245-266.

Török, Z., Vigh, L., and Goloubinoff, P. (1996). Fluorescence detection of symmetric GroEL(14)(GroES(7))(2) heterooligomers involved in protein release during the chaperonin cycle. J. Biol. Chem. 271, 16180-16186.

Hartl, F. U. (1996). Molecular chaperones in cellular protein folding. Nature 381, 571-580.

Azem, A., Diamant, S., Kessel, M., Weiss, C., and Goloubinoff, P. (1995). The protein-folding activity of chaperonins correlates with the symmetric GroEL-14(GroES-7)-2 heterooligomer. Proc. Natl. Acad. Sci. U. S. A. 92, 12021-12025.

Xu, Z., Horwich, A. L., and Sigler, P. B. (1997). The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature 388, 741-750.

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