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.
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.
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 ]
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.
Alternative Conformations of the Loop and the Entropic Penalty for Binding
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
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.
Landry, S. J., Zeilstra-Ryalls, J., Fayet, O., Georgopoulos, C., and Gierasch,
L. M. (1993). Characterization of a Functionally Important Mobile Domain
of GroES. Nature 364, 255-258.
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
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,
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,
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.
End of document