From the Département de Biochimie Médicale, Centre
Médical Universitaire, 1 rue Michel-Servet, 1211 Geneva,
Switzerland and the Department of Biochemistry, Tulane
University School of Medicine, New Orleans, Louisiana 70112
Received for publication, September 20, 2000
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ABSTRACT |
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Chaperonins are universally conserved proteins
that nonspecifically facilitate the folding of a wide spectrum of
proteins. While bacterial GroEL is functionally promiscuous with
various co-chaperonin partners, its human homologue, Hsp60 functions
specifically with its co-chaperonin partner, Hsp10, and not with other
co-chaperonins, such as the bacterial GroES or bacteriophage T4-encoded
Gp31. Co-chaperonin interaction with chaperonin is mediated by the
co-chaperonin mobile loop that folds into a The GroEL (Hsp60; Cpn60) and GroES (Hsp10; Cpn10) families of
molecular chaperones play an essential role in mediating folding of
various substrates (1-3). A great amount of data exists on the
functional and structural properties of Escherichia coli's GroEL and GroES and their homologues. Electron microscopy and x-ray
crystallography revealed that GroEL is a tetradecamer composed of two
rings, stacked back-to-back (4-7). The smaller member, GroES,
functions as a heptamer and binds to either or both ends of GroEL
depending on the nucleotide distribution (8, 9). GroEL displays a weak
ATPase activity that is further reduced in the presence of
co-chaperonin (10, 11). GroEL captures its substrates via its apical
domain, which presents a hydrophobic surface (12). GroES binding to
GroEL through its seven mobile loops locks GroEL into a new
conformation, in which most of the previously exposed hydrophobic
residues of the apical domain now are involved in intersubunit
contacts, leaving the interior wall of GroEL lined with hydrophilic
residues (13). The result is the release of the substrate into the
GroEL-GroES cavity, also referred to as the Anfinsen cage (14). Thus,
an ideal folding environment has been created for the substrate, which
is given ~10 s to attain a folding-competent conformation (3). The
timing is dependent on the rate of ATP hydrolysis (15). GroES discharge is effected by binding of substrate and ATP to the opposite
(trans) ring of GroEL (16-18). Substrate is released in a
folded form or in a form that is competent to fold, or, if it is not
folded, it rebinds to GroEL, at which point the cycle is repeated
(19-21).
Although the above mechanism has been established in large part by
using the E. coli chaperonin system, it probably applies to
most members of the class I chaperonin family (22). However, one
member, the human mitochondrial Cpn60 (Hsp60), differs in its
quaternary structure from the bacterial Cpn60. GroEL oligomerizes as a
tetradecamer, whereas human Hsp60 has been characterized as a heptamer
(23). Such differences in structure have mechanistic implications.
GroEL mutants that form only a single ring bind substrate and GroES
irreversibly (24), presumably because the driving force for substrate
discharge originates from ATP and/or substrate binding to the (missing)
trans ring. If mitochondrial Hsp60 only forms one ring, then
what drives substrate and Cpn10 discharge? This problem was addressed
by Nielsen and Cowan (28), who showed that the mammalian mitochondrial
pair functions by a mechanism that differs at the level of
co-chaperonin and substrate release. While GroEL/ADP/GroES has a
dissociation constant (Kd) in the nanomolar range,
Hsp60/ADP/Hsp10 is such a weak complex that its Kd
could not even be measured.
Still a mystery at the co-chaperonin level is why GroES is excluded
from a productive interaction with Hsp60 while both Hsp10 and GroES
function with GroEL. It has previously been proposed that a
mitochondrial N-terminal acetylation may be necessary to allow
interaction with Hsp60 (25). This hypothesis was disproved when
recombinant mouse Hsp10 was expressed in bacteria, where N-terminal
acetylation does not occur, and the resulting purified protein was
shown to function in conjunction with Hsp60 as well as GroEL (26).
Another conjecture was that the differences of pI could be important in
determining functional specificity, since bacterial GroES is typically
acidic (pI 5.0), while Hsp10 is basic, with a pI close to 9.0 (27).
Since the mobile loop of the co-chaperonin mediates interaction with
the chaperonin, we hypothesized that the specificity may lie in a few
amino acid differences between the GroES and Hsp10 mobile loops.
Conversely, Nielsen and Cowan (28) demonstrated that, by swapping the
co-chaperonin-binding apical domains between GroEL and Hsp60, the
chimeric equatorial-Hsp60-apical-GroEL protein acquired the ability to
interact with GroES.
We have extensively studied the structure and function of the mobile
loops from GroES, Gp31, and Hsp10 co-chaperonins (29-32). The mobile
loop is an unstructured, ~20-residue-long segment that mediates
Cpn10/Cpn60 interaction. Binding captures the mobile loop in a
Here, we show that the essential Cpn60 binding element lies within the
mobile loop sequence of the Hsp10 co-chaperonin. Our studies lead us to
conclude that these sequence variations dictate the affinity
differences between GroES and Hsp10, and thus it is affinity rather
than a distinct conformation that confers Hsp10's preference for Hsp60.
Bacteria and Bacteriophage--
E. coli groEL(E191G)
(originally named groEL44), is isogenic to E. coli B178 (a galE derivative of W3110), which is
sup+, i.e. nonpermissive for
bacteriophage T4 amber (am) mutants (38, 39). For
bacteriophage T4-GT7 transduction experiments, B178( Subcloning of the Human Chaperonin Genes into the pBAD
System--
pBADhsp10 was created by subcloning the
hsp10 cDNA from pJG-10 (31) into the pBAD vector either
alone or with the human chaperonin gene, hsp60, directly
downstream. To subclone in the vector alone, hsp10 was
PCR-amplified using a primer for the 5'-end that contains an
NdeI site and a primer for the 3'-end that contains the
recognition site for EcoRI. For cloning with
hsp60 downstream, hsp10 was amplified by PCR
using the same 5' primer but a different 3' primer containing the
MscI recognition sequence. Plasmid pBADgroEShsp60 was constructed by subcloning the hsp60 gene from
pETcpn60 by cutting with NcoI, blunt-ending, and
then cutting with HindIII. pETcpn60 was a kind
gift from Dr. P. Viitanen (Dupont). The vector pBADgroES has
EcoRI and HindIII sites at the gene's 3'-end.
pBADgroES was first cut with EcoRI, blunt-ended,
and then digested with HindIII. All constructs were tested
for protein overexpression, following transformation into MC1009 bacteria.
Site-directed
Mutagenesis--
pBADgroES(hsp10ml),
pBADhsp10 (ESml),pES(hsp10ml)HisC,
pES(T28P)HisC,
pES(V26M)HisC, pES
(S21T,T28P)HisC, and
pES(S21T,V26M,T28P)HisC were created by the
method of Kunkel et al. (41). The mutations were placed in
plasmid pBADgroES, pBADhsp10, and
pESHisC (a kind gift from Dr. A. Plückthun,
University of Zurich; Ref. 59). The co-chaperonin constructs
were subsequently subcloned into a plasmid containing either the
groEL or the hsp60 gene, thus creating
pBADgroES(hsp10ml)groEL, pBADgroES(hsp10ml)hsp60,
and pBADhsp10(ESml)hsp60. Due to low levels of expression
from pBAD-derived clones expressing hsp10, we changed the
DNA sequence coding the third amino acid from GGA (poorly represented
in E. coli) to GGT (well represented in E. coli)
by using the QuickChangeTM site-directed mutagenesis kit from
Stratagene.
pBADgroES(S21T,V26M,T28P)hsp60 was
also created by introducing mutations in the groES gene by QuickChangeTM mutagenesis.
Transduction and Complementation Experiments--
T4-GT7
transduction experiments were performed as described in Ref. 43 except
that in the donor strain, OF3465, a kind gift from Dr. O. Fayet (CNRS,
Toulouse), the groESgroEL operon is deleted and replaced by
an
To test the growth of
B178( Protein Expression and Purification--
Human Hsp60 was
overexpressed by induction with 1 mM
isopropyl-1-thio-
GroES and GroES(Hsp10ml) were overexpressed in LMG-190 cells, following
transformation by plasmid pgroES and
pgroES(hsp10ml), respectively. Cells were grown to midlog
phase at 37 °C and induced with the addition of 0.05% arabinose.
After 4-5 h of growth, cells were harvested. The purification protocol
used is essentially a procedure described by Richardson et
al. (32) with the following exceptions. For acid precipitation,
following the DEAE-Sepharose column, fractions containing GroES were
precipitated by ammonium sulfate and resuspended in buffer containing
50 mM sodium succinate (pH 4.6). Following overnight
dialysis against the low pH buffer at 4 °C, the precipitated
proteins were removed by centrifugation, and the GroES-containing
supernatant was ammonium sulfate-precipitated. Essentially pure GroES
and GroES(Hsp10ml) protein was obtained following fractionation by HPLC
Superdex G200 gel filtration.
Wild type GroEL was overexpressed from plasmid pBADgroEL
transformed into LMG-190 cells, induced with arabinose, and purified as
described previously (32). Gp31 was purified from pA431
(formerly called pALEX1; Ref. 32) transformed in the MC1009 genetic
background. The purification procedures used were identical to those
described previously (46, 47). The different GroES His-tagged proteins were overproduced from
MC1000
Hsp10 was purified as described by Landry et al. (31). All
proteins were stored at Citrate Synthase Refolding--
The
chaperonin-dependent renaturation of pig heart citrate
synthase (referred to here as citrate synthase) was performed as described previously (32, 48). The following protein concentrations (given for monomers) were used: 4.2 µM GroEL or Hsp60 and
a 4.2 µM concentration (or a fraction of the value as
indicated in Fig. 3A) of co-chaperonins, and 0.2 µM citrate synthase. Citrate synthase at 33 µM was denatured for 30 min at 25 °C in a solution
containing 6 M guanidine hydrochloride, 3 mM
dithiothreitol, and 2 mM EDTA. The refolding buffer
contained 10 mM MgCl2, 2 mM ATP, 1 mM oxaloacetic acid and 20 mM potassium
phosphate, pH 7.4. The co-chaperonin was added last to the refolding
reaction, and its addition indicated the starting time of the reaction.
The refolding reaction was performed at 30 °C and in a total volume
of 400 µl, and citrate synthase activity was measured after 60 min by
measuring the disappearance of acetyl-CoA at 323 nm. For the time-based
measurements, aliquots were taken exactly at times following the
addition of the co-chaperonin to the refolding mix as indicated in Fig.
3B.
GroES Does Not Assist Hsp60 in Refolding Citrate Synthase--
We
have reconfirmed the previously published finding that human
mitochondrial Hsp60 in combination with E. coli GroES is
unable to assist substrate refolding (23). Chemically denatured pig heart citrate synthase was diluted into renaturation buffer
supplemented with ATP and GroELor Hsp60, and either no co-chaperonin,
GroES, Hsp10, or Gp31 was added (Fig. 1).
Citrate synthase is a substrate that requires the assistance of
chaperonin, co-chaperonin, and Mg-ATP (48, 49). We found that GroEL
with any of the three co-chaperonins, GroES, human Hsp10, or
bacteriophage T4 Gp31, assists citrate synthase refolding. In contrast,
Hsp60 helps refold citrate synthase only when partnered with Hsp10.
Neither GroES nor Gp31 increased the levels of refolded citrate
synthase significantly above those of Hsp60 alone, which, in fact,
inhibits citrate synthase refolding. Not surprisingly, Gp31 has closer
functional similarity to GroES than Hsp10 because it interacts with the
same GroEL partner in vivo as GroES. However, GroES and Gp31
share much less sequence similarity (~14%) than GroES and Hsp10
(~44%; Ref. 50; Fig. 2A).
GroES Carrying the Mobile Loop of Hsp10 Assists Hsp60 in Refolding
Citrate Synthase--
Since it has been shown that GroES does not
assist Hsp60 in refolding substrates because GroES does not bind to
Hsp60, then it is likely that elements responsible for the defective
partnership lie within the binding interface of the chaperone proteins
(23). If the mobile loop contains the essential information for binding to Cpn60, then providing the correct mobile loop sequence capable of
recognizing Hsp60 should restore interaction. We constructed the
following two chimeric proteins: GroES containing the Hsp10 mobile
loop, referred to as GroES(Hsp10ml), and Hsp10 with the GroES mobile
loop, referred to as Hsp10(ESml) (Fig. 2C). These proteins
were generated from plasmid gene constructs made by site-directed mutagenesis (for details, see "Experimental Procedures").
We first characterized GroES(Hsp10ml) by purifying the protein and
testing for its ability to assist GroEL or Hsp60 in the refolding of
citrate synthase. The purification properties of GroES(Hsp10ml) were
very similar to those of wild type GroES. The hybrid protein was
functional with GroEL to the same extent as GroES, Gp31, and Hsp10,
yielding approximately an 80% recovery of native citrate synthase
activity. GroES(Hsp10ml) was completely indistinguishable from Hsp10 in
its ability to help Hsp60 in the citrate synthase refolding assays
(Fig. 3). Using different ratios of
co-chaperonin to chaperonin as well as measuring the rates of citrate
synthase refolding, no differences between Hsp10 and GroES(Hsp10ml)
could be detected. These results clearly indicate that all of the
necessary information for co-chaperonin interaction with mitochondrial
Hsp60 lies within the mobile loop sequence.
GroES(Hsp10ml) and Human Hsp60 Can Substitute for GroEL/GroES in E. coli Growth--
Our subsequent approach was to test whether
combinations between human and bacterial chaperonins can substitute for
the bacterial GroEL machinery for E. coli and/or
bacteriophage growth. We used a genetic system to knock out the
chromosomal groESgroEL operon while maintaining viability by
providing the cells with a plasmid carrying the desired chaperonin
genes. OFB3465 is a strain that contains a chloramphenicol
resistance-encoding cassette (CamR) in the place of the
groESgroEL operon (51). Viability of the OFB3465 is
maintained by a plasmid-encoded groESgroEL operon. In
addition, a nearby Tn10 transposon insertion (encoding
tetracycline resistance (TetR)) is 90% cotransducible with
the CamR-encoding cassette. We grew a bacteriophage T4-GT7
lysate on this strain and used it to transduce the B178(
In the absence of any chaperonin genes, such as the pBAD vector alone,
no CamR- resistant colonies were recovered. We tested the
ability of GroES(Hsp10ml) to substitute for GroES for E. coli growth. The hybrid co-chaperonin, when coexpressed along with
E. coli GroEL from plasmid
pBADgroES(hsp10ml)groEL, supports cell viability in the
presence of chloramphenicol; i.e. recipient cells bearing either plasmid exhibit a T4-GT7 cotransduction frequency of 90% (Table
I).
While these experiments were in progress, it was shown that human Hsp60
can substitute for GroEL in E. coli (52). However, Hsp60
requires the presence of Hsp10 and is unable to function in
vivo with GroES. Using the T4-GT7 transduction system, we were able to delete the groESgroEL operon in the presence of
plasmids pBADhsp10hsp60 and
pBADgroES(hsp10ml)hsp60, but not in the presence of
pBADgroEShsp60 (Table I). Contrary to this, we did not
recover any CamR transductants from strains transformed
with plasmid pBADhsp10(ESml)hsp60, providing further
proof that the Hsp10 mobile loop sequence carries the essential
elements that dictate Hsp60 chaperonin binding specificity.
How efficiently do GroES(Hsp10ml) and Hsp60 or GroES(Hsp10ml) and
GroEL function in E. coli? We compared the colony-forming units of cells bearing the deletion of the endogenous
groESgroEL operon and carrying either
pBADgroES(hsp10ml)groEL or
pBADgroES(hsp10ml)hsp60. Cells carrying plasmid-encoded
GroEL grow better at higher temperatures, compared with cells carrying
plasmid-encoded Hsp60 (Fig. 4). We also
examined the ability of both strains to support bacteriophage growth.
We found that only bacteriophage T5 efficiently formed plaques when
GroEL was replaced by Hsp60. Bacteriophage GroES(Hsp10ml) Possesses a Higher Affinity for
GroEL(E191G)--
We hypothesized that sequence alterations in the
mobile loop affect co-chaperonin interaction with Cpn60 function by
changing the structure and dynamics of the mobile loop. Using NMR
techniques, Landry et al. (54) showed that the mobile loop
of Hsp10 is less dynamic than that of GroES. Thus, it may exhibit a
greater affinity than GroES for GroEL. Likewise, GroES(Hsp10ml) should
exhibit a higher affinity than GroES for GroEL.
To test this hypothesis, we transformed a low affinity GroEL mutant
strain, groEL(E191G), with plasmids expressing either GroES
or GroES(Hsp10ml) (32). Bacteriophage A GroES Mutant Protein Carrying Three Amino Acid Substitutions in
Its Mobile Loop Can Functionally Interact with Hsp60--
If a high
affinity co-chaperonin is sufficient to create functional interaction
with Hsp60, then key residues in the Hsp10 mobile loop might be
identified by potentially large individual contributions to high
affinity. The amino acid sequences of the GroES and Hsp10 mobile loops
are similar, albeit with a few significant differences (Fig.
2B). These sequence differences affect two aspects of the
mobile loop that regulate its affinity for Cpn60. First, the sequence
of the hydrophobic tripeptide that makes direct contact with Cpn60
should be more hydrophobic in Hsp10 than GroES. Hsp10's middle
tripeptide residue, methionine, is more hydrophobic than valine (53)
found at the equivalent position in GroES. Second, the amino acids at
two positions in Hsp10, which regulate the balance between mobile loop
flexibility and a preference toward the chaperonin-bound conformation,
favor the chaperonin-bound conformation more than the residues in the
GroES sequence that occupy the equivalent positions. A distinguishing
feature is the presence of a highly conserved proline residue following
the hydrophobic tripeptide in mitochondrial mobile loops. A Pro residue
at this position should reduce the conformational dynamics of the
mobile loop, thus making the Hsp10 mobile loop less flexible than its bacterial counterpart. Indeed, it is known that mutations at this position, P33S or P33H, reduce the affinity of yeast Hsp10 for GroEL
(33, 34). Most likely, substitution at this site results in increased
conformational dynamics at position 33, which in turn increases the
entropic cost of binding to GroEL (31, 54). Finally, the nature of the
residue at position 21 in GroES and the corresponding position 26 in
Hsp10 is important in determining
To test whether these three mobile loop features are necessary and
sufficient to specify Hsp60 interaction, we created the following GroES
mutants: GroES(S21T,V26M,T28P)HisC, GroES(S21T,T28P)HisC, GroES(V26M)HisC, and GroES(T28P)HisC. The proteins were expressed with C-terminal histidine (His tags) and purified from strains expressing Gp31 instead of wild type GroES. Because Gp31 and GroES do
not form mixed oligomers, we were able to obtain pure, homogenous His-tagged mutant proteins. As controls, we also purified wild type
GroES and GroES(Hsp10ml), each with C-terminal His-tags. All six
proteins were tested for their ability to assist either GroEL or Hsp60
in refolding citrate synthase. All co-chaperonins when paired with
GroEL helped recover ~80% of the denatured citrate synthase (Fig.
5). As already shown with the
non-His-tagged proteins, GroES (with a His tag) and Hsp60 provided no
assistance for renaturation of citrate synthase, while the use of the
His-tagged version of GroES(Hsp10ml) and Hsp60 resulted in ~80%
renaturation of citrate synthase. Neither of the single point mutants,
GroES(V26M) or GroES(T28P), were functional with Hsp60 in the refolding
assay. Interestingly, the double mutant, GroES(S21T,T28P), helped
recover partial citrate synthase activity, while the triple mutant,
GroES(S21T,V26M,T28P), was almost as active as Hsp10 and
GroES(Hsp10ml) in its partnership with Hsp60 in assisting citrate
synthase refolding. Therefore, it is likely that all three mobile loop
substitutions are necessary to bestow an Hsp10-specific activity.
The Mutant GroES Protein Can Only Partially Support Bacteriophage
It is possible that this in vivo assay is far more sensitive
than the in vitro citrate synthase refolding assay.
Therefore, to discern subtle functional differences between the entire
Hsp10 mobile loop and that of GroES with the S21T, V26M, and T28P
changes, we created the construct
pBADgroES(S21T,V26M, T28P)hsp60 and tested its ability to
support E. coli growth (Table I). Compared with pBADhsp10hsp60 and pBADgroES(hsp10ml)hsp60,
pBADgroES(S21T,V26M,T28P)hsp60 functions equally well for
supporting E. coli growth in the absence of the endogenous
groES and groEL genes.
Our understanding of the mechanism of chaperonin-assisted folding
has greatly increased over recent years, yet there are many details
that remain unresolved or under debate. For example, it is well
accepted that the regulation of the timing of co-chaperonin binding to
chaperonin depends on nucleotide hydrolysis (15). On the other hand,
the co-chaperonin plays a critical role in controlling both the
efficiency and specificity of chaperonin-assisted folding. Beyond
playing a structural role in the chaperone machine, the co-chaperonin
also acts as an allosteric modulator (56). We have previously proposed
that the GroEL-binding co-chaperonin mobile loop has evolved a finely
tuned balance between flexibility and conformational preference for a
3:5 Before dissecting the various features of the mobile loop, we first set
out to identify the essential and specific co-chaperonin feature in
Hsp10 that distinguishes it from GroES in its ability to interact with
Hsp60. Because GroES does not bind to Hsp60, we hypothesized that it is
the mobile loop that contains the essential information for binding to
chaperonin. Therefore, providing GroES with the correct mobile loop
sequence capable of recognizing Hsp60 should restore interaction.
Indeed, the mobile loop of Hsp10 grafted into GroES,
resulting in GroES(Hsp10ml), is sufficient to provide functional
specificity. Furthermore, GroES(Hsp10ml) is able to function in
vivo for growth of E. coli and bacteriophage T5 when partnered with Hsp60.
From our indirect affinity measurements of the mobile loops, we
concluded that the Hsp10 mobile loop sequence restored interaction between GroES and Hsp60 because the Hsp10 mobile loop increases GroES
affinity. This observation is in agreement with previously published
NMR data showing that the mobile loop of Hsp10 is less flexible than
that of GroES (54).
What are the specific features of the mobile loop sequence that
determine co-chaperonin affinity? We approached this question by
mutational analysis and found that three substitutions in the GroES
mobile loop are necessary and sufficient to acquire Hsp10-like specificity, as summarized in Fig. 6.
These three residues most likely provide a combination of increased
hydrophobicity and reduced flexibility in the Hsp10 mobile loop,
explaining why Hsp10 exhibits higher chaperonin affinity than GroES.
Since the single ring Hsp60 obviously cannot utilize binding of GroES
and ATP to the trans ring as a driving force for the
discharge of cis GroES, the single ring chaperonin may have
evolved an intrinsically lower affinity for co-chaperonin. The high
affinity mobile loop of Hsp10 may compensate for an intrinsically low
co-chaperonin affinity in single ring Hsp60 (28).
-hairpin conformation
upon binding to the chaperonin. A delicate balance of flexibility and
conformational preferences of the mobile loop determines co-chaperonin
affinity for chaperonin. Here, we show that the ability of Hsp10, but
not GroES, to interact specifically with Hsp60 lies within the mobile loop sequence. Using mutational analysis, we show that three
substitutions in the GroES mobile loop are necessary and sufficient to
acquire Hsp10-like specificity. Two of these substitutions are
predicted to preorganize the
-hairpin turn and one to increase the
hydrophobicity of the GroEL-binding site. Together, they result in a
GroES that binds chaperonins with higher affinity. It seems likely that
the single ring mitochondrial Hsp60 exhibits intrinsically lower
affinity for the co-chaperonin that can be compensated for by a higher affinity mobile loop.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hairpin conformation, which presents three highly conserved
hydrophobic residues at the binding interface. Both flexibility and the
GroEL-bound
-hairpin structure are conserved among co-chaperonins.
Mutations encoding substitutions throughout the GroES and Hsp10 mobile
loops have been identified that alter affinity by affecting the
flexibility of the mobile loop (33-37). Mutants that allow better
binding are probably sequence alterations that disfavor mobility, while
mutants that weaken binding either increase the entropy, and therefore
flexibility of the mobile loop, or promote an incorrect mobile loop
conformation (32).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), which carries
a
prophage, thus making it less susceptible to T4-GT7 infection,
was used as the recipient strain. DH5
supE and CJ236 were
used for cloning and site-directed mutagenesis purposes (40, 41).
Bacteriophages
, T5, T4, RB49, RB43, and T4 31amNG71
(carries an amber allele in gene 31; Refs. 35 and 42) are
from our collection, that of H. Krisch at the University of Toulouse,
or that of R. H. Epstein at the University of Geneva.
-chloramphenicol resistance (CamR) cassette.
Transductants were selected on medium containing 50 µg/ml ampicillin
or 20 µg/ml kanamycin, 12 µg/ml tetracycline, and 0.02%
L-arabinose and then scored for CamR.
)
groESgroEL:CamR × pBADgroES(hsp10ml)groEL versus × pBAD
groES(hsp10ml)hsp60 (Fig. 4), single colony isolates were
resuspended in LB medium, and 10-fold serial dilutions were spot-tested
on LB plates supplemented with 50 µg/ml ampicillin, 15 µg/ml
chloramphenicol, and 0.1% arabinose and incubated for 24-36 h at the
temperatures indicated in Fig. 4. Bacteriophage growth was tested by
spot testing aliquots of serial dilutions of various bacteriophage on
LB plates containing 50 µg/ml ampicillin, 15 µg/ml chloramphenicol,
and 0.1% arabinose and seeded with 0.2 ml of culture grown for 24 h in LB medium containing 50 µg/ml ampicillin, 15 µg/ml
chloramphenicol, and 0.02% arabinose. Plates were incubated for
20 h at 37 °C. Complementation experiments were performed as
described previously (44).
-D-galactopyranoside from the pET
vectors transformed in BL21. Transformants were grown to midlog phase
and induced for 3 h before harvesting. Hsp60 was purified exactly
as described in Ref. 45.
groESgroEL:CamR(pK631groEL)
transformed with the ampicillin-resistant plasmids: pESHisC,
pES (hsp10ml)HisC,
pES(T28P)HisC,
pES(V26M)HisC,
pES(S21T,T28P)HisC, or
pES(S21T,V26M,T28P)HisC. Because this strain
expresses Gp31 instead of GroES, pure mutant GroES protein was obtained
(Gp31 and GroES do not formed mixed
oligomers).1 The strains were
grown in LB medium supplemented with 15 µg/ml chloramphenicol, 20 µg/ml kanamycin, 50 µg/ml ampicillin, and 0.02% arabinose to
midlog phase and induced with 1 mM
isopropyl-1-thio-
-D-galactopyranoside. After ~3 h of
growth at 37 °C, cells were harvested. Purification procedures were
identical to those described in protocols 8 and 12 of Ref. 58. for
Ni2+-nitrilotriacetic acid Superflow 5-ml columns.
Purification was performed at either room temperature or 4 °C (see
Fig. 7 for gel from purification steps of wild type His-tagged GroES
and His-tagged GroES(V26M)HisC).
80 °C in a buffer containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, and 15% (v/v) glycerol. Protein
concentrations were measured by either absorption at 280 nm using molar
extinction coefficients determined by quantitative amino acid analysis
or by the Bradford protein assay method, standardized with known
concentrations of either GroEL, GroES, or Gp31.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Hsp60 assists the refolding of citrate
synthase only with help from Hsp10 and not GroES or Gp31. The
yield of folded protein is expressed as a percentage of nondenatured
citrate synthase activity. Citrate synthase activity was measured after
60 min of refolding at 25 °C with the indicated combinations of
chaperones, as described under "Experimental Procedures." Data
presented are the average of three separate experiments, with the S.E.
indicated.
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Fig. 2.
Sequence comparisons and creation of chimeric
co-chaperonins. A, alignment of E. coli
GroES, human Hsp10, and bacteriophage T4 Gp31 co-chaperonin sequences.
B, multiple sequence alignment of selected bacterial
versus mitochondrial co-chaperonin mobile loops. The
GroEL-binding hydrophobic tripeptide is highlighted in
purple; residues that are important in regulating mobile
loop flexibility are colored green and blue.
C, hybrids between different co-chaperonin core sequences
and mobile loop sequences, generated by site-directed
mutagenesis.
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Fig. 3.
A hybrid protein between GroES and the Hsp10
mobile loop (GroES(Hsp10ml)) is completely indistinguishable from Hsp10
in its ability to help Hsp60 in the refolding assays.
A, comparison of Hsp10 and GroES(Hsp10ml) in combination
with Hsp60 by testing for citrate synthase refolding activity by using
different ratios of co-chaperonin to chaperonin. B,
comparison of Hsp10 and GroES(Hsp10ml) in combination with Hsp60 by
testing for citrate synthase refolding activity by measuring the rates
of refolding.
) recipient
strain, transformed with various chaperonin plasmid constructs. We
first selected for the inheritance of the TetR marker. The
number of TetR transductants is indicative of the
transduction efficiency, since inheritance of the Tn10
transposon alone does not produce a defective phenotype. We then scored
for coinheritance of the CamR cassette. Inheritance of the
CamR marker is only possible if the genes provided in
trans can completely substitute for groESgroEL in
bacterial viability. A 90% coinheritance is expected when the
plasmid-encoded genes are able to functionally substitute for the
chromosomal groESgroEL genes, as was observed when the
recipient strain carried the pBADgroESgroEL plasmid (Table I).
Deletion of the groESgroEL operon by T4-GT7-mediated transduction in
the presence of different combinations of human and bacterial
chaperonins
exhibited a reduced
efficiency of plaque formation on a strain expressing Hsp60, and
bacteriophages T4 and its distant relative RB49 were unable to form
plaques on this strain because Gp31 does not interact with Hsp60, in
agreement with previously published results (52).
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Fig. 4.
Hsp60 maintains E. coli and
bacteriophage viability, although to a reduced extent compared with
endogenous GroEL. A, plating properties of 10-fold
dilutions of single isolates of
B178( )
groESgroEL:CamR × pBADhsp10hsp60 (1),
B178(
)
groESgroEL:CamR × pBADgroES(S21T,V26M,T28P)hsp60 (2),
B178(
)
groESgroEL:CamR × pBADgroES(hsp10ml)hsp60 (3), and
B178(
)
groESgroEL: CamR × pBADgroES(hsp10ml)groEL (4) at different
temperatures. B, spot various dilutions of bacteriophages
T5, T4,
, RB43, and RB49 on bacterial lawns as indicated in the
figure. Plates were incubated for 20 h at
37 °C.
does not form plaques on
groEL(E191G) (38). Overexpression of GroES did not overcome
the block on bacteriophage
; however, overexpression of Hsp10 or
GroES(Hsp10ml) in groEL(E191G) allowed growth of
bacteriophage
(Table II). This result
suggests that Hsp10 and GroES(Hsp10ml) restore interaction with the low
affinity GroEL(E191G) enough to assist bacteriophage
maturation.
Bacteriophage growth on various bacterial mutant constructs
on groEL(E191G)
mutant bacteria transformed with plasmids expressing GroES or various
mutant proteins as indicated in the first column. Plaque-forming units
are expressed in comparison with that on B178 wild type bacteria (taken
as 1.0). Lawns were created by mixing 0.2 ml of a fresh overnight
culture with soft agar. LB-agar plates contained ampicillin and either
0.05% arabinose (rows 1-4) or 5 mM
isopropyl-1-thio-
-D-galactopyranoside (rows 5-11) and
were incubated for 20 h at 37 °C.
-sheet propensity. Threonine, as
found in the Hsp10 sequence, exhibits a higher
-sheet propensity
than serine (55) at the equivalent site in GroES (32). The three
features described above, differentiating the Hsp10 and GroES mobile
loops, support the idea that Hsp10 has evolved to possess a higher
affinity mobile loop.
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Fig. 5.
Chaperonin-assisted refolding of citrate
synthase. All three changes, S21T, V26M, and T28P, are required
for efficient functional interaction of GroES with Hsp60 in this assay.
The double mutant, GroES(S21T,T28P)HisC helps partially, while
the single mutants, GroES(V26M)HisC and GroES(T28P)HisC, do not
function with Hsp60. Data presented are the average of three separate
experiments, with the S.E. indicated.
Growth--
Do the mobile loops of GroES(S21T,V26M,T28P),
GroES(Hsp10ml), and Hsp10 differ from that of wild type GroES simply
because they have higher affinity for chaperonin; or is the mechanism more complicated, involving both affinity differences and a more specific conformational adaptation necessary for interaction with Hsp60? As described above, the co-chaperonin affinity can be
qualitatively measured using an in vivo complementation
assay. The low affinity groEL(E191G) mutant strain was
transformed with the following plasmids: pESHisC,
pES(hsp10ml)HisC, pES(T28P)HisC,
pES(V26M)HisC, pES(S21T,T28P)HisC, and
pgroES(S21T,V26M,T28P)HisC, and protein expression was induced with
isopropyl-1-thio-
-D-galactopyranoside. The ability of
bacteriophage
to form plaques was measured at 37 °C (Table II).
Transformants expressing the single mutant GroES(T28P)HisC allowed
formation of medium size plaques, although transformants expressing
GroES(V26M)HisC or GroES(S21T, T28P)HisC behaved like wild type
GroESHisC. GroES(S21T,V26M,T28P)HisC and GroES(T28P)HisC behaved
equally in partially suppressing
growth at higher temperatures, in
contrast to their differing abilities to interact with Hsp60 in
vitro. Furthermore, only GroES(Hsp10ml)HisC was able to
efficiently support
growth at all temperatures. Although this assay
does not measure affinity directly, it demonstrates that the GroES triple mutant lacks a component necessary for completely restoring interaction with GroEL(E191G) at least at higher temperatures.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hairpin loop that is optimized for continued cycles of binding
and release (30, 32). Here, we have shown that co-chaperonin affinity
for GroEL is dictated by a balance between disorder and structure of
the mobile loop, as well as the hydrophobicity of the conserved
internal tripeptide, which constitutes the actual binding site.
View larger version (41K):
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Fig. 6.
GroES assumes Hsp10 specificity with three
amino acid substitutions in the region of the mobile loop that forms
a hairpin upon binding to GroEL. Each
substitution in GroES(S21T,V26M,T28P) results in higher chaperonin
affinity. Threonine has the highest propensity for
-sheet structure
of any amino acid at a solvent-exposed site (green).
NMR experiments suggest that a proline at this site reduces the
conformational flexibility of the mobile loop (red). The
hydrophobic tripeptide makes direct contact with GroEL
(purple). Met is more hydrophobic than Val. The illustrated
structure corresponds to the GroEL-bound mobile loop peptide
conformation (PDB:1EGS) determined by trNOE NMR spectroscopy (30),
except the N-terminal NH2 is replaced with threonine.
We propose that the difference in affinity between Hsp60 and GroEL is achieved by altered domain-domain interactions within Hsp60, very likely analogous to the substitution that reduces co-chaperonin-affinity in GroEL(E191G) (32). Thus, the actual co-chaperonin-binding sites of GroEL and Hsp60 could be nearly identical, which would explain the simultaneous increase in affinity for both Hsp60 and GroEL(E191G) obtained by the S21T, V26M, and T28P substitutions in GroES. Nevertheless, Hsp60 function may require tighter binding than would be obtained with a GroES-like mobile loop; thus, the Hsp10 mobile loop has acquired a higher affinity for chaperonins through the incorporation of these residues in the native sequence.
Increased demands on the chaperonin machine imposed by bacteriophage
have helped to reveal the importance of a self-consistent mobile
loop structure. The GroES(Hsp10ml)-GroEL(E191G) combination can support
growth, but the GroES(S21T,V26M,T28P)-GroEL(E191G) combination is
less efficient. Perhaps the chaperonin affinity of the GroES triple
mutant is still not quite correct. Previously, we have shown that
chaperonin affinity must be delicately balanced to support growth of
bacteriophage T4 (32); thus, this combination of substitutions may
produce an affinity below or above the level required for optimal
chaperonin function. In either case, other residues in the GroES or
Hsp10 mobile loops could further modulate the affinity toward the
appropriate level. Alternatively, appropriate chaperonin affinity is
not sufficient for optimal function, and the internal workings of the
mobile loop are under additional constraints, such as might be required
for an important intermediate in the chaperonin reaction cycle. Such
sophisticated constraints could explain the unique requirement for the
GroES homolog, Gp31, in the assembly of bacteriophage T4 Gp23 into
capsids (57). In contrast, chaperonin function for cellular growth
either is less constrained or more easily compensated by the other
chaperone systems.
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ACKNOWLEDGEMENTS |
---|
We thank F. Keppel for help with the transduction experiments and J. Guidry and K. Steede for purification of Hsp10. We thank P. Viitanen, A. Plückthun, O. Fayet, and F. Krisch for the generous gifts of plasmids, bacterial strains, and bacteriophage.
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FOOTNOTES |
---|
* This work was supported by Swiss National Foundation Grant 31-47283.96, the canton of Geneva (to C. G.), and National Science Foundation Grant MCB-9512711 (to S. J. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 41-22-702- 55-12; Fax: 41-22-702-55-02; E-mail: Costa.Georgopoulos@ medecine.unige.ch.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M008628200
1 A. Richardson, F. Schwager, S. J. Landry, and C. Georgopoulos, unpublished observation.
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