(Received for publication, August 15, 1994; and in revised form, October 29, 1994)
From the
The DnaK (Hsp70), DnaJ, and GrpE heat shock proteins of Escherichia coli constitute a cellular chaperone system for protein folding. Substrate interactions are controlled by the ATPase activity of DnaK which itself is regulated by the nucleotide exchange factor GrpE. To understand the structure-function relationship of this chaperone system, the quaternary structures of DnaK, GrpE, and DnaK-GrpE complexes were analyzed by gel filtration chromatography, dynamic light scattering, analytical ultracentrifugation, and native gel electrophoresis. GrpE formed dimers in solution. DnaK formed monomers, dimers, and higher mole mass oligomers, the equilibrium between these forms being dependent on the DnaK concentration. The behavior of DnaK and GrpE in gel filtration and dynamic light scattering suggested elongated shapes of both molecules. In the absence of added nucleotides, DnaK and GrpE formed stable complexes containing one molecule of DnaK and two molecules of GrpE. A 44-kDa N-terminal ATPase fragment of DnaK also formed complexes with GrpE with the same 1:2 stoichiometry. DnaK-GrpE complex formation was unaffected by elimination of DnaK-bound nucleotides or addition of saturating concentrations of a DnaK peptide substrate. These findings allow the correlation of DnaK-GrpE interactions with a role for GrpE in the functional cycle of the DnaK chaperone system.
Hsp70 stress proteins (Hsp70) are molecular chaperones that
assist folding and degradation of proteins as well as disassembly of
protein complexes(1) . A key feature of their chaperone
function is the ability of Hsp70 to bind nonnative protein substrates
and to release them upon ATP binding(2) . ()The
ATPase activity of at least some Hsp70 proteins is under tight control
of cofactors allowing coordination of ATP binding/hydrolysis and
substrate interaction. For the Escherichia coli homologue
DnaK, its low intrinsic ATPase activity is jointly stimulated by the
DnaJ and GrpE heat shock proteins acting at different steps of the
reaction(3) . In particular, GrpE is a nucleotide exchange
factor of DnaK (3, 4) and, apparently as a consequence
of this activity, mediates the efficient release of DnaK-bound
substrates thereby allowing recycling of DnaK(5) . GrpE is
essential for chaperone activities of DnaK in vivo(4) and in vitro(5, 6) .
Limited information exists on the quaternary structures of DnaK,
DnaJ, and GrpE, and the complexes formed between them(1) . DnaK
was found to form monomers and a minor fraction of dimers in gel
filtration chromatography and monomers in glycerol gradient
sedimentation(7) . GrpE has a relative mole mass (mole mass) of
22 kDa and was suggested to form monomers based on glycerol gradient
sedimentation analysis(8) . DnaK and GrpE interact both in
vivo and in vitro(9) . DnaK-GrpE complexes are
stable in the absence of added nucleotides and are disrupted by
Mg/ATP(8) . A loop structure exposed at the
surface of the N-terminal ATPase domain of DnaK is essential for stable
GrpE binding(10) .
To elucidate the relation of structure and function of the DnaK system we analyzed in detail the quaternary structures of DnaK, GrpE, and DnaK-GrpE complexes with a variety of biochemical and biophysical methods. The hydrodynamic parameters determined in the present study offer new insight into the quaternary structures of these proteins and the role of DnaK-GrpE interactions in the functional cycle of the DnaK chaperone system.
Figure 1:
SDS-PAGE (A) and Superose 12
gel filtration analysis (B) of GrpE and DnaK. A: lane 1, mixture of standard proteins (soybean trypsin
inhibitor, 21.5 kDa; carbonic anhydrase, 31 kDa; porcine lactate
dehydrogenase, 36.5 kDa; bovine glutamic dehydrogenase, 55.4 kDa;
bovine serum albumin, 66.3 kDa); lane 2, DnaK (1.5 µg); lane 3, GrpE (1.5 µg). B, elution profiles of
standard proteins (horse myoglobin, 17 kDa; chicken ovalbumin, 44 kDa;
and bovine gamma globulin, 158 kDa; V indicates
the position of the void volume), GrpE (69 µg), and DnaK (69
µg). Roman numerals I, II, and III label
different peaks of the DnaK chromatogram. Vertical dashed lines are drawn in the positions of some peaks for convenient
comparisons.
The 44-kDa ATPase
fragment of DnaK (DnaK1-385) was overproduced in E. coli cells (strain MC4100) carrying pUHE21-2fd12 (dnaK1-385) (13) upon
isopropyl-1-thio-
-D-galactopyranoside induction of
expression of the plasmid-encoded gene and was purified as described (10) .
Preparation of nucleotide-free DnaK was done by J.
Reinstein and will be described elsewhere. ()Protein
concentrations were determined by colorimetric assays (BCA protein
assay; Pierce) and calibrated by amino acid analysis.
Purified DnaK and GrpE were fully active by several criteria, including chaperone-mediated refolding of firefly luciferase (DnaK and GrpE), ATPase activity (DnaK and DnaK1-385), and substrate binding (DnaK)(6, 13) .
where k is the Boltzmann constant, T the
temperature, and the viscosity of the solvent (
=
1.017 mPa s at 20 °C). For a globular protein of mass M the radius R can be estimated from the following
equation,
where v is the partial specific volume (v = 0.735 cm/g), h the hydration of typical proteins (h = 1.3), and N
Avogadro's number.
From the observed diffusion coefficients D obtained in light scattering and the sedimentation velocities s the mole masses were calculated using the following Svedberg equation,
where R is the gas constant and the density of
the buffer (
= 0.999 g/cm
).
Nondenaturing PAGE (8% acrylamide) was performed as described(10) , and the relative amounts of GrpE, DnaK, and GrpE-DnaK complexes were determined using a Hirschmann Elscript 400 scanner.
In
quasi-elastic light scattering (QLS) experiments, the correlation
functions obtained indicate a slightly broader size distribution of
GrpE than expected for a strictly monodisperse sample of globular
protein. When the data were fitted to a single exponential, diffusion
coefficients of 4.8-5.6 10
cm
s
were obtained for different preparations of
GrpE at a concentration of 0.6 mg/ml (Table 1).
Sedimentation velocity centrifugation of GrpE at the concentration used for QLS experiments yielded a single band with a velocity s of 2.7 S (Table 1). Knowledge of the s value as well as the diffusion coefficient determined by QLS permitted usage of the Svedberg equation to calculate a mole mass for GrpE of 44-51 kDa. Sedimentation equilibrium centrifugation of GrpE at the same concentration yielded mole masses for GrpE of 38.0 and 40.2 kDa (Table 1).
Figure 2: Influence of DnaK concentration on gel filtration profile. A DnaK stock solution (16 mg/ml) was diluted at 20 °C to different end concentrations as indicated by the labels of the profiles in mg/ml. Aliquots of 50 µl of the stock solution and the dilutions were subjected to Superose 12 column chromatography. The reduction of optical densities caused by the dilution factor (relative to the stock solution) was compensated for by a corresponding amplification of the monitor signal. The integral of the total profile is therefore almost constant in every chromatogram. The vertical dashed line in the position of DnaK peak III indicates that the positions of corresponding peaks remain constant in all chromatograms.
It is likely that the different forms of DnaK are in equilibrium with each other. To test this possibility, we fractionated the eluate of a gel filtration run with DnaK injected into the Superose 12 column at a concentration of 16 mg/ml. The DnaK concentrations in fractions containing the peak maxima were estimated to be more than 100-fold lower (<0.1 mg/ml) than in the injected sample. After equilibration for about 3 h at room temperature, fractions containing the maxima of peaks I, II, and III were injected into the column. The elution profiles showed mainly a single peak at the position of the original peak III (Fig. 3). Analysis of peak I showed an additional minor peak at the position of former peak II. This additional peak was not observed when the material was equilibrated overnight before analysis (data not shown).
Figure 3: Chromatography of different DnaK peaks. The chromatogram of a DnaK sample at 16 mg/ml (labeled ``DnaK'') yielding peaks I, II, and III, and the chromatograms of materials obtained from these peaks after equilibration for 3 h at concentrations of less than 0.1 mg/ml are shown.
In QLS analysis, DnaK at
a concentration of 1 mg/ml showed a broad size distribution yielding
diffusion coefficients from 3.4 to 4.3 10
cm
s
. In sedimentation velocity
centrifugation, DnaK yielded two bands of 4.0 and 6.3 S (Table 1). When these two sedimentation velocities were combined
with the upper and lower limits of the diffusion coefficients observed
in QLS, mole masses of 85 and 165 kDa were calculated using the
Svedberg equation. The DnaK profile observed in equilibrium
centrifugation did not fit with a single exponential. When fitted with
two exponentials, mole masses of 67 and 180 kDa were obtained with
optical density contributions of 75 and 25%, respectively (Table 1). Furthermore, the data fitted well with a model
assuming coexistence of monomers, dimers and trimers with weight
percentages of 75, 12, and 14%, respectively.
Figure 4: Gel filtration analysis of DnaK-GrpE mixtures at different molar ratios. Samples of 50 µl at 20 °C containing 2 nmol of GrpE (GrpE), 1 nmol of DnaK (DnaK), or mixtures of 1 nmol of DnaK and 0.5 nmol (1:0.5), 1 nmol (1:1), 2 nmol (1:2), 3 nmol (1:3), and 4 nmol (1:4) of GrpE were injected into a Superose 12 column. The monitor signal was attenuated 2-fold in some cases (1:2, 1:3, and 1:4) leading to a 2-fold reduction of peak sizes. Vertical dashed lines are drawn to facilitate comparisons of DnaK peak I (K(I)), complex peak (C), GrpE peak (E) and DnaK peak III (K(III)).
For QLS and
analytical ultracentrifugation analyses, DnaK and GrpE were mixed at a
molar ratio of 1:2 (1.0 mg/ml DnaK and 0.63 mg/ml GrpE) and
equilibrated overnight prior to the analyses. In QLS, the size
distribution of the mixture was more narrow than that of DnaK alone and
yielded a diffusion coefficient of 3.7 10
cm
s
(Table 1). In
sedimentation velocity centrifugation, the proteins exhibited a single
migrating band with 4.8 S. Using the Svedberg equation we calculated a
mole mass of the DnaK-GrpE complex of 117 kDa. In sedimentation
equilibrium centrifugation, the equilibrium profile of the complex was
fitted to a single exponential, corresponding to a mole mass of 116 kDa
(121 kDa, respectively, for an independent second experiment).
Figure 5:
Kinetics of DnaK-GrpE complex formation.
Samples of 50 µl containing DnaK (0.75 nmol) and GrpE (1.5 nmol)
were injected at 1 min (1m), 16 min (16m), 46 min (46m) or 15 h (15h) after mixing at 20 °C into a
Superose 12 column, running at a flow rate of 1 ml/min. The obtained
profiles in the peak region are superimposed. V indicates the position of the column void volume (identical with
the position of DnaK peak I).
Interestingly, although the oligomeric forms of DnaK (peaks I and II) were slowly converted by GrpE to DnaK-GrpE complexes, they had a limited capability of binding GrpE, as revealed by the following experiment. DnaK and GrpE were mixed at a molar ratio of 1:2 but, in order to increase the fraction of oligomeric DnaK, on ice and at higher concentrations (9.2 mg/ml DnaK and 5.8 mg/ml GrpE). After 1 min the mixture was injected into a Superose 12 column operated at high flow rate (1 ml/min), and the eluted fractions were subjected to SDS-PAGE. Coomassie-stained GrpE and DnaK bands were quantified by densitometry, and their ratios were plotted against fraction numbers. GrpE was also detected in fractions containing oligomeric DnaK (Fig. 6A). The molar ratio DnaK/GrpE decreased with increasing fraction numbers proportional to elution volumes and approximated the value 1:2 in the region of peak C (Fig. 6B).
Figure 6: The distribution of DnaK and GrpE within the eluate of a fast gel filtration run (1 ml/min) of a DnaK-GrpE mixture at a molar ratio of 1:2 (DnaK:GrpE), injected 1 min after mixing at 0 °C, was analyzed by SDS-PAGE (A) and densitometry (B). A, lane 1 (label ``M''), mixture of standard proteins (see Fig. 1A); lanes 2-15, aliquots of fractions of the eluate of the gel filtration run. Lanes are labeled by the fraction numbers. B, ratios of integrated DnaK and GrpE bands of different lanes of gel A are plotted against their positions within the elution profiles represented by the fraction numbers. Maxima of DnaK peak I (K(I)), complex peak (C), GrpE peak (E) and DnaK peak III (K(III)) were found in fractions 2-3, 15, 19, and 21, respectively, as indicated by the arrows. Band integration ratios corresponding to molar DnaK/GrpE ratios of 1:1 and 1:2 were estimated by reference gels and are indicated by horizontal lines.
Figure 7:
Titration DnaK and DnaK1-385 with
GrpE. Full-length DnaK (12 µg; upper) and DnaK1-385
(8 µg; lower) were incubated for 30 min at 30 °C with
amounts of GrpE ranging from 0- to 4-fold molar excess with respect to
DnaK/DnaK1-385. The mixtures were analyzed by native PAGE and the
relative amounts of free DnaK () and GrpE-complexed DnaK (
)
in each sample were determined by densitometry and are expressed as
percent of total DnaK.
The effects of the presence of DnaK substrates on
GrpE binding were tested by pre incubating DnaK with saturating amounts
of the well characterized DnaK/Hsp70 substrate peptide C of vesicular
stomatitis virus glycoprotein(17) . This 13 -mer peptide binds
to DnaK with a K of 2.6
µM(13) . GrpE was added at a 2-fold molar excess
with respect to DnaK. After incubation of the samples for 30 min at 30
°C, they were loaded onto a Superdex 200 column equilibrated in 20
µM peptide C containing running buffer. The obtained
chromatogram was not different compared with that obtained from an
experiment omitting peptide C during the preincubation of DnaK and in
the running buffer, indicating that GrpE-DnaK complexes formed with the
same efficiency and characteristics in the presence as in the absence
of peptide substrate (chromatograms not shown).
Biochemical and biophysical analyses of the quaternary structures of GrpE and DnaK identified GrpE as a dimer and DnaK as a mixture of monomers, dimers, and higher oligomers which are in a slow equilibrium. DnaK oligomers were dissociated in the presence of GrpE. Furthermore, DnaK-GrpE complexes were shown to consist of one molecule DnaK and two molecules GrpE and to be formed independently of the presence of peptide substrates or DnaK-bound nucleotides. These results provide novel insight into the structural and functional relationships of this chaperone system.
The sedimentation coefficient that we determined (s = 2.7) is in good
agreement with the sedimentation coefficient for GrpE obtained from
glycerol gradient centrifugation (s
=
2.5); however, these data were interpreted by assuming a globular shape
for GrpE (calibration with globular reference proteins) and,
consequently, a monomeric structure for GrpE was proposed(8) .
Our results clearly indicate a nonglobular, possibly elongated, shape
of GrpE
, since it elutes in gel filtration as a 190-kDa
species, its diffusion coefficient is higher and sedimentation velocity
is smaller than expected for globular proteins of 44 kDa, and the
dissociated molecule migrates slower than expected in SDS-PAGE.
Finally, our result agrees with results from glutaraldehyde
cross-linking experiments ( (25) as reviewed by Georgopoulos et al.(19) ).
The low peak elution volumes
relative to globular reference proteins and the low diffusion
coefficients suggested elongated shapes for DnaK molecules. In contrast
to the GrpE peak, however, DnaK peaks II and III were
significantly shifted to higher elution volumes when gel filtration was
performed in the presence of high salt concentrations, indicating a
more flexible structure for DnaK than for GrpE
.
The different forms of DnaK are in a slow equilibrium for which oligomerization is favored upon increasing the concentration of DnaK. Similar concentration-dependent distributions of oligomeric states of DnaK were found in size exclusion HPLC and nondenaturing gel electrophoresis(20) . DnaK oligomers obtained after gel filtration were quantitatively converted to monomers as shown by chromatography. This conversion resulted from establishment of the equilibrium after removal of the monomers and dilution of DnaK by a factor of more than one-hundred during the experiment. The time required for establishment of the equilibrium is long (>1 h) compared with the time needed for fast gel filtration of DnaK (less than 12 min).
Our
experiments identify the GrpE dimer as the functionally active species.
Its mode of binding to the ATPase domain of a DnaK monomer remains
unclear. A GrpE dimer might have a single DnaK binding site or,
alternatively, two binding sites which, however, cannot bind two DnaK
molecules simultaneously with high affinity as we did not observe
DnaK-GrpE
complexes at 1:1 stoichiometry.
We suggest that monomeric DnaK is the active molecule in
DnaK-mediated protein folding. Oligomeric DnaK would constitute a pool
of latent chaperone being activated during heat stress (temperature
increase, increasing amounts of unfolded protein, increase of
GrpE expression(19) ).
We showed that GrpE also has affinity for a DnaK-substrate complex. This result leads
us to suggest that GrpE
plays a role not only in the named
housekeeping of DnaK monomers but also in subsequent interactions
involving DnaK; much evidence suggests that this is related to the
nucleotide binding by DnaK. GrpE accelerates the release of ATP or ADP
bound to DnaK(3) . Our data show that GrpE
was
bound to the ATPase domain of DnaK, regardless of whether nucleotide
(ADP) was prebound to DnaK or peptide substrate was present. It is
therefore possible that GrpE
binds to DnaK-substrate
complexes and dissociates DnaK-bound nucleotides (ADP), functioning as
a nucleotide exchange factor. Although GrpE
remains bound
to the DnaK-substrate complexes rebinding of ATP might then induce
dissociation of GrpE
and substrate from DnaK.
Whether
the observed stoichiometry of the DnaK-GrpE complex has any
implications for protein folding in vitro and the role of DnaJ
are subjects of our present investigations.