Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1996 Dec 24;93(26):15024-9.
doi: 10.1073/pnas.93.26.15024.

Chaperone activity and structure of monomeric polypeptide binding domains of GroEL

R Zahn  1 , A M Buckle, S Perrett, C M Johnson, F J Corrales, R Golbik, A R Fersht
Affiliations

Chaperone activity and structure of monomeric polypeptide binding domains of GroEL

R Zahn et al. Proc Natl Acad Sci U S A. .

Abstract

The chaperonin GroEL is a large complex composed of 14 identical 57-kDa subunits that requires ATP and GroES for some of its activities. We find that a monomeric polypeptide corresponding to residues 191 to 345 has the activity of the tetradecamer both in facilitating the refolding of rhodanese and cyclophilin A in the absence of ATP and in catalyzing the unfolding of native barnase. Its crystal structure, solved at 2.5 A resolution, shows a well-ordered domain with the same fold as in intact GroEL. We have thus isolated the active site of the complex allosteric molecular chaperone, which functions as a "minichaperone." This has mechanistic implications: the presence of a central cavity in the GroEL complex is not essential for those representative activities in vitro, and neither are the allosteric properties. The function of the allosteric behavior on the binding of GroES and ATP must be to regulate the affinity of the protein for its various substrates in vivo, where the cavity may also be required for special functions.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Cloning of the apical domain of GroEL and various of its fragments. The utilized expression vector coded for an N-terminal histidine-tail (ht) composed of 36 amino acids and containing a thrombin cleavage site (vertical arrow). Alternatively, a shorter version of this histidine-tail (sht) containing 17 amino acids was used. The N and C termini of the generated fusion proteins, namely ht-GroEL191-298, ht-GroEL191-322, ht-GroEL191-328, ht-GroEL191-337, ht-GroEL191-345, ht-GroEL191-376, sht-GroEL191-345, and sht-GroEL191-376 are indicated by horizontal arrows. Secondary structure is indicated by boxes and arrows for α-helix (shaded) or 310-helix (open) and β-sheet structure, respectively. Assignment of secondary structure of residues 191 to 336 was from the crystal structure of sht-GroEL191-345 using procheck (22) and the algorithm of Kabsch and Sander (23). Numbering of α-helices and secondary assignment of residues 337 to 376 according to Braig et al. (3).
Figure 2
Figure 2
Catalysis of amide proton exchange of barnase (2.4 mM) by the fragments GroEL191-345 (a) and sht-GroEL191-376 (b). The results are very similar to those described in refs. and for intact GroEL. The rate constants (in units of min−1) for the exchange of individual NH protons in barnase in the presence of fragment [formula image (+G)] are plotted against those in the absence (formula image). Amide protons that exchange by global, mixed, and local unfolding mechanisms are displayed by circles, triangles, and squares, respectively. The plot for 90 μM sht-GroEL191-345 at pD 7.1 (not shown) is virtually superimposable on that for sht-GroEL191-376 (b). (pD = pH measured in 2H2O.) It is clearly seen that those protons that require global unfolding for exchange have significantly increased rates, thus showing that the fragments bind to the unfolded state of barnase and catalyze its unfolding. We could only estimate the final concentration of GroEL fragment in a since GroEL191-345 tended to crystallize during the exchange experiment at the high initial protein concentration.
Figure 3
Figure 3
Refolding of rhodanese and cyclophilin A in the presence of sht-GroEL191-345 and sht-GroEL191-376. (a) Relative enzymatic activity of rhodanese (0.1 μM) after refolding in the presence (+) or absence (−) of GroEL (2.5 μM monomer), GroES (2.5 μM monomer), ATP (2 mM), sht-GroEL191-345 (2.5 μM), sht-GroEL191-376 (2.5 μM), or bovine serum albumin (45 μg/ml), from 8 M urea (U). One-hundred percent activity was obtained with native rhodanese (N). (b) Refolding kinetics of rhodanese in presence of GroEL, GroES, and ATP. The final concentrations are the same as in a. One-hundred percent activity was obtained with native rhodanese. (c and d) Refolding kinetics of rhodanese in the presence of 0.18 μM, 2.5 μM, or 5 μM sht-GroEL191-345 and sht-GroEL191-376, respectively. (e) Refolding of 1 μM cyclophilin A in the presence of 7 μM GroEL (monomer), 4 μM sht-GroEL191-345, 4 μM sht-GroEL191-376, or 1 μM sht-GroEL191-376. One-hundred percent activity was obtained with native cyclophilin A. Standard error bars are shown. The 30% spontaneous refolding of cyclophilin was complete in the dead time of the experiment.
Figure 4
Figure 4
Thermal denaturation of sht-GroEL191-376 (upper trace) and of sht-GroEL191-345 (lower trace). (a) Monitored by far UV-CD at 222 nm. (b) Monitored by differential scanning calorimetry.
Figure 5
Figure 5
The three-dimensional structure of sht-GroEL191-345. Secondary structure representation is drawn with molscript (37) and raster3d (38). Helices are labeled as in Braig et al. (3). N and C refer to the N terminus (residue 191) and C terminus (residue 336) of the model, respectively. The backbone representation (Upper Right) is in the same orientation as Upper Left, color-coded according to B factor of main-chain atoms: blue (20 Å2) to red (60 Å2), drawn with program o (27). (Lower) Representative region of electron density, calculated using refined coordinates, viewed along the helices H8 and H9.
See this image and copyright information in PMC

Comment in

  • Dissecting intrinsic chaperonin activity.
    Clore GM, Gronenborn AM. Clore GM, et al. Proc Natl Acad Sci U S A. 1997 Jan 7;94(1):7-8. doi: 10.1073/pnas.94.1.7. Proc Natl Acad Sci U S A. 1997. PMID: 8990150 Free PMC article. No abstract available.

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ellis R J, Hartl F U. FASEB J. 1996;10:20–26. - PubMed
    1. Braig K, Otwinowski Z, Hegde R, Boisvert D C, Joachimiak A, Horwich A L, Sigler P B. Nature (London) 1994;371:578–586. - PubMed
    1. Braig K, Adams P D, Brünger A T. Nat Struct Biol. 1995;2:1083–1094. - PubMed
    1. Fenton W A, Kashi Y, Furtak K, Horwich A L. Nature (London) 1994;371:614–619. - PubMed
    1. Chen S, Roseman A M, Hunter A S, Wood S P, Burston S G, Ranson N A, Clarke A R, Saibil H R. Nature (London) 1994;371:261–264. - PubMed

Publication types

MeSH terms

LinkOut - more resources