From the Département de Biochimie
Médicale, University of Geneva, 1 rue Michel-Servet, 1211 Geneva,
Switzerland and
Department of Biochemistry, Tulane University
School of Medicine, New Orleans, Louisiana 70112
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ABSTRACT |
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Previous work has shown that the GroEL-GroES
interaction is primarily mediated by the GroES mobile loop. In
bacteriophage T4 infection, GroES is substituted by the gene
31-encoded cochaperonin, Gp31. Using a genetic selection
scheme, we have identified a new set of mutations in gene
31 that affect interaction with GroEL; all mutations result
in changes in the mobile loop of Gp31. Biochemical analyses reveal that
the mobile loop mutations alter the affinity between Gp31 and GroEL,
most likely by modulating the stability of the GroEL-bound hairpin
conformation of the mobile loop. Surprisingly, mutations in
groEL that display allele-specific interactions with mutations in gene 31 alter residues in the GroEL
intermediate domain, distantly located from the mobile loop binding
site. The observed patterns of genetic and biochemical interaction
between GroES or Gp31 and GroEL point to a mechanism of genetic allele specificity based on compensatory changes in affinity of the
protein-protein interaction. Mutations studied in this work indirectly
alter affinity by modulating a folding transition in the Gp31 mobile
loop or by modulating a hinged conformational change in GroEL.
Chaperonin-assisted folding of certain substrates depends on the
coordinated interaction of GroEL, ATP, and GroES (1, 2). Certain
unfolded or partially folded polypeptides bind to GroEL, a
double-toroid, tetradecameric protein composed of 58-kDa subunits arranged with 7-fold symmetry (3-5). GroES, made up of 10.5-kDa subunits arranged in 7-fold symmetry (6, 7), binds to GroEL, thus
stabilizing a conformational change that doubles the
substrate-containing cavity of GroEL and promoting the release of the
substrate into the cavity (8, 9). The amount of time the substrate
spends in the cavity depends on the rate of ATP hydrolysis in the GroEL cis ring and the release of GroES (10, 11). In turn, the
release of GroES is promoted by the binding of ATP or ATP and GroES to the trans GroEL ring (11, 12). After GroES release, the
polypeptide substrate is released either in a folded or a
folding-competent state or in a conformation still recognizable by
GroEL, in which case it binds to the same or a different GroEL molecule
(13). The efficient cycling of the GroE chaperone machine is essential to ensure that the chaperonin can provide the necessary folding assistance to its substrates (14-16).
GroEL is essential for bacteriophage T4 growth. The mutant
groEL44(E191G) allele has been shown to block bacteriophage
T4 growth at the level of capsid head assembly, i.e. in
groEL44(E191G) mutant cells, Gp23, the major capsid protein,
aggregates into amorphous lumps (17). The same phenotype was previously
observed during infection of a wild type host by a bacteriophage T4
defective in gene 31 (18). Subsequent analyses of genetic
suppressors identified an interaction between the host groEL
gene and the bacteriophage T4 gene 31 (19).
It turned out that Gp31 is functionally analogous to GroES despite low
sequence identity (14% at the amino acid level) (20, 21), and it can
completely replace GroES for Escherichia coli growth.1 The crystal
structures of GroES and Gp31 reveal significant structural identities
as expected from their similar in vivo and in
vitro function (6, 22). Both GroES and Gp31 subunits bear a
flexible polypeptide segment, identified by nuclear magnetic resonance (NMR) spectroscopy and limited proteolysis (23, 24). The mobile loops
mediate GroEL-GroES binding through a central hydrophobic tripeptide
(Ile25-Val26-Leu27) as shown by NMR
studies and confirmed by the crystal structure of the
GroEL·ADP·GroES complex (8, 23).
All mutations identified thus far in either groES or gene
31, which result in defective GroEL interaction, alter amino
acid residues in the mobile loop. Interestingly, most of these GroES substitutions do not affect the IVL tripeptide. Rather, the best characterized groES mutant alleles affect either of the two
glycine residues preceding the IVL residues (25) and that participate in formation of a Mutations in groEL, originally identified on the basis of
blocking bacteriophage growth, affect residues that are distant from
the mobile-loop binding site. The affected residues lie in the
intermediate domain that links the ATP binding equatorial domain with
the substrate and GroES binding apical domain (23). The
GroEL·ADP·GroES crystal structure revealed that the intermediate segment, in fact, provides two hinges that allow for the large en
bloc movements in GroEL, which are captured by GroES binding (8).
Genetic analyses have revealed that groEL mutations
fall into two classes on the basis of their allele-specific interaction with groES (25, 27) or gene 31 (17) mutations.
The study concerning GroEL-Gp31 interaction revealed that of two
groEL mutants that block bacteriophage Understanding how specific mutations in these genes affect chaperone
function is the overall aim of the work described in this paper. Taking
advantage of a simple genetic selection system, we have identified new
mutations in gene 31 of bacteriophage T4 that
switch the phenotype from suppression of groEL44(E191G) to suppression of groEL515(A383T). Biochemical analyses reveal
that these gene 31 mutations exert their effects primarily
by altering the affinity of Gp31 for GroEL.
Genetic Selection--
Twenty independent lysates of the
original T4 Cloning of Wild Type and Mutant Genes--
Mutant gene
31(L35I, T31A) was amplified by polymerase chain reaction;
the polymerase chain reaction fragment was cloned into the
EcoRI and XbaI sites of the high copy pBAD vector
pMPM201 (29), and the resulting clone overproducing Gp31(L35I,T31A) was
named pALEX5. The wild type gene 31 was cloned in the same manner, except the amplification was done from a wild type T4 plaque
isolated on a B178 lawn, and the resulting clone was named pALEX1. The
Gp31(I36W) protein was overproduced from pALEX32, created by the
introduction of the corresponding mutation, resulting in I36W by
site-directed mutagenesis (30) using pALEX1 as the template. Mutant
gene 31 (L35I) was made by site-directed mutagenesis (30)
using the plasmid pSV25 (wild type gene 31) as the template (20). All clones were sequenced in their entirety using either the
standard Sanger sequencing method or automated sequencing (Li-CORE).
pBADgroESgroEL is a plasmid that expresses the wild type
groES and groEL genes under the control of the
arabinose-induced promoter. The groES and groEL
genes were cloned as a 2.1-kilobase pair fragment from pOFx62(27)
containing 45 base pairs upstream of the starting ATG codon. The
fragment was cloned into pBAD22 (31).
Plasmid pBADgroESgroEL(A383T), used to overexpress
GroEL(A383T), was constructed by replacing the
BstXI-SmaI fragment from pBADgroESgroEL with the corresponding
BstXI-SmaI fragment from plasmid pOF1153 (27).
The authenticity of the clone was verified by sequencing 300 base pairs
around the altered codon.
Protein Purification--
Wild type GroEL and GroES were
overexpressed from pBADgroESgroEL transformed into MC1009
cells and induced with arabinose and purified essentially as described
previously (23). Residual peptides bound to GroEL were removed by
Affi-Gel Blue chromatography in the presence of buffer containing 50 mM Tris-HCl, pH 7.5, 125 mM NaCl, 1 mM ATP, and 2.5 mM MgCl2 followed
by MonoQ chromatography to remove GroEL-associated nucleotide (buffer:
20 mM Bis-Tris, pH 6.0, 50 mM-1M
KCl gradient).
GroEL(A383T) was overproduced from pBADgroESgroEL(A383T)
transformed into groEL515(A383T) cells, and GroEL(E191G) was
overproduced from pJZ548(27) transformed into the
groEL44(E191G) mutant background. Both mutant proteins were
purified following the same procedure as that used for wild type GroEL.
Gp31, Gp31(L35I), Gp31(L35I,T31A), and Gp31(I36W) were purified from
the overexpressing plasmids described above, all in the MC1009 genetic
background. The purification procedures used were identical to those
previously described (20). The expected molecular mass of wild type
Gp31, Gp31(L35I,T31A), and Gp31(I36W) were confirmed by electrospray
mass spectroscopy.
All proteins were stored at Citrate Synthase Refolding--
The
chaperonin-dependent renaturation of pig heart citrate
synthase (referred to in the text as citrate synthase) was performed as
described previously (32). The following protein concentrations (given
for monomers) were used: 4.2 µM chaperonins and
cochaperonins and 0.2 µM citrate synthase. Citrate
synthase at 33 µM was denatured for 30-60 min at
27 °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 refolding
reaction was performed at 27 °C in a total volume of either 200 µl
or 400 µl, and citrate synthase activity was measured after 60 min.
Complex Formation--
Complex formation was initiated by adding
ATP to a final concentration of 1 mM to a solution
containing 50 mM Tris-HCl, pH 7.7, 7.8 mM
MgCl2, 1 mM KCl, 1 mM
dithiothreitol, 8.4 µM GroEL, and 35.2 µM
Gp31 (monomers). The reaction mixture (250 µl) was left at 22 °C
for 10 min. An aliquot of 200 µl was loaded onto a TSK 3000G
gel-fitration column that had been equilibrated in 50 mM
Tris-HCl, pH 7.7, 10 mM MgCl2, 1 mM
KCl, 0.01% (w/v) Tween 20, and 0.23 mM ATP. The column was
run at 22 °C at a flow rate of 1 ml/min. The fraction between 10.5 and 11.5 min was collected. Samples were acetone-precipitated and
analyzed by means of electrophoresis on a 15% polyacrylamide gel
containing SDS. The proteins were stained with Coomassie Brilliant Blue.
Fluorescence Experiments--
A Photon Technologies Inc. Quanta
Master luminescence spectrometer with a double excitation set-up and
gloved cuvette holder maintained at 25 °C with a cooler-heater water
bath system was used for fluorescence analysis. All reactions were
performed under constant stirring and in a total volume of 2 ml. The
intensity at 337 nm was monitered as a function of time with excitation at 295 nm and all slits adjusted to 4 nm. The following protein concentrations were used (in monomers): 2.0 µM GroEL, 1.4 µM Gp31(I36W), and 1.4 µM Gp31 and its
mutant variants. ATP was used at 1 mM with a buffer
containing 100 mM Tris-HCl, pH 7.5, 1 mM
MgCl2, and 1 mM dithiothreitol. Data were
initially analyzed in Felix software provided with the spectrometer and
converted into a Kaleidagraph for presentation. The background
fluorescence intensity contribution associated with
nontryptophan-containing proteins (expressed as a percentage of the
fluorescence intensity for Gp31(I36W) alone)) was subtracted
accordingly: GroEL, 20%; Gp31, 8%, Gp31(L35I), 5%; Gp31(L35I,T31A),
6%.
Transferred Nuclear Overhauser Effect
(trNOE)2 NMR
Analysis--
Carboxamide peptides corresponding to the mobile loops
of Gp31, Gp31(L35I), and Gp31(L35I,T31A) were synthesized using
9-fluorenylmethoxycarbonyl chemistry, acetylated off-line, and purified
by reverse-phase high preformance liquid chromatography. Sequences are
as follows: Gp31, AQAGDEEVTESGLIIGKRVQ; GP31(L35I),
AQAGDEEVTESGIIIGKRVQ; GP31(L35I,T31A), AQAGDEEVAESGIIIGKRVQ.
Peptide sequences were confirmed by matrix-assisted laser
desorption ionization mass spectrometry (MALDI). GroEL and GroEL(E191G)
were exchanged and concentrated into a 50 mM potassium
phosphate, pH 6.1, buffer (Centricon-30, Amicon). GroEL was added to a
final concentration of 60 µM in an NMR sample containing
2 mM peptide, 10% D2O, and 0.3 mM
trimethylsilyl proprionate in 50 mM potassium phosphate, pH
6.1, buffer. Spectra were recorded at 30 °C on a General Electric OMEGA PSG 500 NMR spectrometer operating at 500.05 MHz frequency. Data
were processed as described previously (24) using Felix software
(Biosym Technologies, San Diego, CA) running on a Silicon Graphics
Indigo (Mountain View, CA) work station.
groEL/Gene 31 Allele-specific Mutations Fall into Two
Classes--
Previous work has shown that bacteriophage T4 mutations,
which restore growth on groEL44(E191G) map to gene
31 (17), and sequencing of one candidate, called T4 Mutations in groEL and Gene 31 Affect Chaperonin-assisted Refolding
of Citrate Synthase--
Previous work has shown that citrate synthase
depends on both GroEL and GroES for renaturation (32). In the absence
of chaperonins, only 10-20% of denatured citrate synthase regains
activity. GroEL alone (with ATP) inhibits refolding of citrate synthase
(Fig. 1). In contrast, GroEL paired with
GroES, Gp31, Gp31(L35I), or Gp31(L35I, T31A) efficiently helps citrate
synthase refolding. Likewise, GroEL(E191G) inhibits refolding of the
substrate in the absence of a cochaperonin but assists citrate synthase
refolding with GroES. However, when paired with Gp31, GroEL(E191G) is
unable to assist refolding. As anticipated from our in vivo
genetic analysis, Gp31(L35I) restores chaperonin-assisted folding by
GroEL(E191G), whereas Gp31(L35I,T31A) and GroEL(E191G) form a
nonfunctional pair for the refolding of citrate synthase.
The above results indicate that citrate synthase refolding depends on
functional interaction between GroEL and Gp31, strongly suggesting that
the observed defects in bacteriophage T4 growth are also the result of
aberrant chaperone-assisted folding. What is the molecular basis of the
defect in chaperonin-cochaperonin interaction? The amino acid
substitutions in Gp31 or GroEL could increase or decrease their
affinity for each other. To test this, we analyzed complex formation by
gel filtration chromatography.
The L35I Substitution in Gp31 Restores Binding to
GroEL(E191G)--
Formation of the GroEL·Gp31 complex requires
nucleotide and can be observed by size fractionation on gel filtration
chromatography (Fig. 2A). With
the same conditions, GroEL(E191G) does not form a complex with Gp31
(Fig. 2B). Therefore, the inability of GroEL(E191G) to
assist citrate synthase refolding when paired with Gp31 is most likely
because of a lack of stable chaperonin-cochaperonin complex formation.
However, GroEL(E191G)·ATP does form a stable complex with Gp31(L35I)
(Fig. 2C). Thus, we conclude that the substitution in
Gp31(L35I) increases the affinity of Gp31 affinity for GroEL, and we
hypothesize that Gp31(L35I,T31A) decreases the affinity of Gp31 for
GroEL. To test this, we developed a technique to distinguish subtle
differences in GroEL binding between Gp31 and Gp31 mutants.
Relative GroEL-binding Affinities of Gp31 Mutants Determined by
Competition with a Fluorescent Gp31 Variant--
Tryptophan
fluorescence may be exploited to measure protein-protein interactions
provided that the tryptophan undergoes an environmental change upon
formation of the complex. Because neither GroEL, GroES, nor Gp31
contain tryptophan residues, we sought to introduce one such that it
would report Gp31 binding to GroEL. Gp31(I36W) was created by
site-directed mutagenesis. Ile36 is the central residue of
the hydrophobic tripeptide,
Leu35-Ile36-Ile37, in the Gp31
mobile loop. Gp31(I36W) interacts with GroEL in a manner similar to
wild type Gp31, consistent with the fact that it can substitute for
wild type Gp31 in bacteriophage T4 growth. However, unlike wild type
Gp31, Gp31(I36W) functions with GroEL(E191G) in citrate synthase
refolding (Fig. 1). Nevertheless, Gp31(I36W) can be exploited as a
reporter to detect the relative binding affinities of the various GroES
and Gp31 proteins in a competition assay.
The fluorescence emission spectrum of Gp31(I36W) exhibits a wavelength
of maximum emission (
The relative binding affinities of Gp31 and Gp31 mutants can be
evaluated with a binding competition assay. First, a complex between
GroEL and Gp31 is formed in the presence of nucleotide. Subsequently,
binding of Gp31(I36W) to GroEL is monitored by tryptophan fluorescence.
The extent that Gp31 blocks the change in fluorescence indicates the
ability of the Gp31 competitor to inhibit Gp31(I36W) binding.
Prior incubation of GroEL·ATP with Gp31 only slightly inhibits
Gp31(I36W) binding (Fig. 3B). In contrast, Gp31(L35I)
complexed with GroEL·ATP effectively blocks Gp31(I36W) binding,
indicating that it is more difficult to displace than its wild type
counterpart. Gp31(L35I,T31A) in complex with GroEL·ATP hinders
Gp31(I36W) binding partially, neither as well as Gp31(L35I) nor as
weakly as Gp31. From these experiments, the following relative order of
cochaperonin binding affinity to GroEL·ATP is established: Gp31(L35I) > Gp31(L35I, T31A) > Gp31.
Because the affected residues in the GroEL mutants are distant from the
mobile loop binding site, one would expect the same order of binding
affinity on the GroEL mutants used in this study. We tested this
prediction by repeating the same experiment while substituting GroEL
with GroEL(A383T). Indeed, the order of relative affinity remains the
same (data not shown).
Synthetic Mobile Loop Peptides Corresponding to Gp31 Mutants
Recapitulate Altered GroEL Binding--
Above we showed that mutations
that affect residues in the mobile loop of Gp31 alter its affinity for
GroEL. To establish that these differences are a direct result of
changes in the mobile loop binding to GroEL, we compared the GroEL
binding properties of synthetic peptides corresponding to the mobile
loops of our Gp31 mutants by analysis of trNOEs. The appearance of
trNOEs in the NOESY spectra of the three peptides in the presence of
GroEL indicates that each binds to GroEL (Fig.
4). However, the Gp31(L35I) peptide
exhibits more intense trNOEs compared with those of Gp31 and
Gp31(L35I,T31A) peptides, suggesting that the L35I substitution strengthens mobile loop binding to GroEL and that the T31A substitution weakens it. In the presence of GroEL(E191G), there is a marked decrease
in trNOEs for Gp31 and Gp31(L35I,T31A) compared with Gp31(L35I),
consistent with the lack of in vivo interaction between GroEL(E191G) and these cochaperonins. These results indicate that the
mobile loop itself can recognize the defect in GroEL(E191G) despite the
relatively large distance between the altered amino acid residue and
the mobile loop binding site in GroEL (Fig.
5A).
GroEL-Gp31 Mutant-Suppressor Pairs Illustrate a Mechanism of
Allele-specific Genetic Interaction--
As stated earlier, taking
advantage of a GroEL mutant, GroEL(A383T), that does not function with
Gp31(L35I), we have isolated compensatory mutations that reveal a
striking genetic interaction between Gp31 and GroEL. Specifically, all
mutants of bacteriophage T4 Gp31(L35I) isolated as restoring ability to
grow on groEL515(A383T) simultaneously lose their ability to
grow on groEL44(E191G). Potentially, the GroEL-Gp31 genetic
interaction could be ascribed to a conventional mechanism of allele
specificity, in which distinct mutant-suppressor pairs arise from
direct contacts among the affected amino acids in the protein-protein
interface (33). However, amino acids involved in the GroEL-Gp31 genetic
interactions analyzed here are located far from each other in the
GroEL·Gp31 complex (Fig. 5A).
Our biochemical analyses of the mutant Gp31 proteins suggest that
mutant-suppressor pairs complement each other by contributing in
opposite ways to GroEL-Gp31 affinity. The relative affinities of wild
type and mutant Gp31 proteins were probed indirectly through their
ability to block binding to GroEL of a fluorescent variant of Gp31,
Gp31(I36W), and by the strength of trNOEs observed for their
corresponding mobile loop peptides in the presence of GroEL. Both
assays indicate the following relative affinity for GroEL: Gp31(L35I) > Gp31(L35I,T31A) > Gp31. Thus, it appears that the increased GroEL
binding affinity of Gp31(L35I) compensates for the low affinity
interaction of the GroEL(E191G)-Gp31 pair, and the reduced affinity of
Gp31(L35I,T31A) compensates for the presumed high affinity interaction
of the GroEL(A383T)-Gp31(L35I) pair. The same relative affinity ranking
is observed for binding of the various Gp31 proteins to GroEL(A383T)
(data not shown) and for binding of the corresponding mobile loop
peptides to GroEL(E191G). The fact that the order of affinity is
indifferent to the GroEL protein tested supports our proposal that the
mechanism of GroEL-Gp31 genetic interactions studied here arises from
compensatory affinity changes rather than classical allele-specific
alterations in the structure of the binding interface.
Amino Acid Substitutions in the Mobile Loop Affect Formation of the
GroEL-bound Hairpin Conformation--
As detailed above, the
predominance of substitutions at amino acid position 31 in the
pseudorevertants (14 of 20 isolates; Table I) suggests that changes at
this position have a greater potential for counteracting the increased
binding affinity caused by the L35I substitution. Consistent with this
hypothesis, previous NMR studies detected trNOEs between the side
chains of Thr31 and Leu35 in the Gp31 mobile
loop peptide bound to GroEL, indicating that these side chains approach
within a few angstroms of each other (24). The proposed
Thr31-Leu35 contact corresponds to the
Ser21-Ile25 contact observed in the GroEL-bound
GroES mobile loop peptide (23). All of the trNOE data in the Gp31
peptide were consistent with its forming the same 3:5 hairpin
conformation (using the classification of Sibanda and Thornton (34)) as
that formed by the corresponding GroES mobile loop peptide (Fig.
5B), which places these two side chains next to each other
but on opposite strands of the hairpin.
Amino acid
Our experimental results, coupled with the conclusions derived from the
model systems discussed above, demonstrate that the GroEL-Gp31 genetic
interaction can be understood in the framework of a folding transition
by the Gp31 mobile loop. The mobile loop conformational dynamics must
be exquisitely poised for folding into a Amino Acid Substitutions in the GroEL Hinge Regions Affect Local as
Well as Large Scale Conformational Changes--
Substitutions in the
hinges could change the distribution of GroEL subunits between apical
domain-open and apical domain-closed conformations and/or affect the
apical domain-mobile loop docking interaction by allosteric
communication. We compared the relative orientation of the three GroEL
apical domain residues that contact the mobile loop in the crystal
structure of GroEL·ADP·GroES with the orientation of these residues
in the crystal structure of GroEL alone (Fig.
6 (8, 36)). The orientation of the
Val264 side chain is shifted with respect to other apical
domain residues, suggesting that the Implications for Protein-Protein Interaction--
An
affinity-based mechanism for allele specificity has been ascribed to
other protein-protein interactions, raising the possibility that the
classical notion of allele-specificity is generally avoided by robust,
flexible protein-protein contacts. For example, suppression of defects
in fimbrin-actin interactions in yeast has been attributed to a global
increase in affinity of fimbrin mutants for actin mutants, and at least
two fimbrin mutants bind more tightly to wild type actin (37). Crystal
structures of human fimbrin and bovine actin reveal that residues
affected in the mutant yeast proteins are not only localized to
surfaces of potential protein-protein contact, and several are buried
in a hydrophobic core (38). In the bacterial chemotaxis system,
interaction of the response regulator CheY with the receptor kinase
CheA has been localized to a surface of CheY, but residues in the
contact surface are not evolutionarily conserved, and crystal
structures of CheY with the P2 domain of CheA reveal at least three
different modes of binding (39, 40). In the chaperonin system, we find
that mutations affecting GroEL-Gp31 affinity modulate a folding
transition in the Gp31 mobile loop. Hence, in all of these systems,
residues in the intermolecular interfaces may be less critical than
residues controlling domain folding and stability.
Strict allele-specificity may always involve a conformational switch in
one partner of a protein-protein interacting pair. In the chemotaxis
system, bias for clockwise versus counter-clockwise motor
rotation is controlled by interactions of phospho-CheY with the
flagellar switch protein FliM. Many, if not all mutations in FliM that
suppress mutations in CheY are thought to adjust the bias of the switch
rather than restore normal interactions with mutant CheY (41, 42). The
indirect effect of these suppressor mutations in FliM is analogous to
the effect of hinge mutations in GroEL that compensate for strong or
weak binding by mobile loop mutants in Gp31. Apparently, a great deal
of redundancy has accumulated in the structural features of
protein-protein interactions, as has already been appreciated in
protein folding itself (43). Mutations tend to shift conformational
equilibria between broad energy minima rather than cause distinct
changes in structure.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
-hairpin turn (24). Bacteriophage T4 gene 31 mutant alleles affect a number of residues in the mobile
loop including residue Leu35, corresponding to
Ile25 in the GroES mobile loop (26).
growth,
groEL44(E191G) and groEL515(A383T), only the
former blocks bacteriophage T4 growth. Surprisingly, mutations in gene
31, that restore bacteriophage T4 growth on groEL44(E191G), simultaneously prevent plaque formation
on groEL515(A383T).
EXPERIMENTAL PROCEDURES
1(Gp31(L35I)) mutant were plated separately on
groEL515(A383T) bacteria. Plaque formers, occurring at a
frequency of approximately 10
6 were isolated, restreaked,
and characterized for plating ability on different groEL
mutant hosts. The minimal gene 31 was amplified by
polymerase chain reaction using Dynazyme Taq polymerase from a plaque isolated from a groEL515(A383T) lawn, and the
polymerase chain reaction product was sequenced directly using the
Amersham Pharmacia Biotech Delta Taq sequencing kit (28).
All 20 suppressors sequenced contained the original
1 mutation
(L35I) and in addition had a mutation that altered a second amino acid
residue, also localized in the mobile loop. Twelve of these
pseudorevertants had a mutation that resulted in a change at codon 31 of Thr to Ala, and two candidates had a change at the same site from a
Thr to an Ile.
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 or Gp31.
RESULTS
1,
showed that the mutation in gene 31 results in an amino acid
change at codon 35 from Leu to Ile (26). This apparently subtle change
in Gp31 resulted in a strikingly different genetic interaction with
GroEL, because T4
1 simultaneously lost its ability to plaque on
groEL515(A383T) mutant bacteria. We took advantage of the
finding that T4
1 does not grow on groEL515(A383T) mutant
bacteria to isolate spontaneously occurring, bacteriophage-encoded
suppressors. From 20 independent T4
1 lysates plated on
groEL515(A383T), bacteriophage "revertants" capable of
forming a plaque were isolated at a frequency of approximately 10
6. All revertants simultaneously lost their capacity to
propagate on groEL44(E191G) mutant bacteria. Sequence
analysis showed that all 20 candidates retain the original
1
mutation (Leu to Ile at codon 35). The most frequently occurring
suppressor mutation (12 of 20 isolates) resulted in a substitution at
codon 31 of Thr with Ala, and two additional revertants were shown to
alter the same codon to an Ile (Table I).
Significantly, the genetic interaction between residues 35 and 31 in
the mobile loop of Gp31 coincides with the physical interaction
observed in the GroEL-bound conformation determined by trNOE NMR (Fig.
5B) (24).
Plating ability of various bacteriophage T4 mutant strains at 37 °C
. However, at a
frequency of 10
6-10
7, spontaneously occurring
revertants can be isolated as plaque-formers on the various
nonpermissive bacterial hosts.
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Fig. 1.
Chaperonin-dependent refolding of
citrate synthase. 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 27 °C with the
indicated combinations of chaperones, as described under
"Experimental Procedures." Data presented are the average and S.E.
of three separate experiments.
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Fig. 2.
Complex formation of mutant and wild type
GroEL and Gp31 proteins. A Commassie Brilliant Blue-stained gel is
presented showing the proteins present in the GroEL-containing fraction
following chromatography on a TSK 3000G column. Lanes
represent mixtures containing: A, GroEL and Gp31;
B, GroEL(E191G) and Gp31; C, GroEL(E191G) and
Gp31(L35I). The asterisk (*) indicates either wild type or
mutant protein.
max) of 347 nm, which suggests that
the introduced tryptophan side chain is solvent-exposed (Fig. 3A). The addition of GroEL in
the absence of nucleotide results in a small increase in fluorescence
intensity and no change in emission
max. However,
further addition of ATP results in a 2-fold increase in emission
intensity as well as a 10-nm decrease in
max. These
results suggest that the tryptophan side chain is transferred to a
nonpolar environment in the GroEL·Gp31 complex.
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Fig. 3.
Inhibition of Gp31(I36W) binding to GroEL by
Gp31 mutant proteins monitored by fluorescence. A,
fluorescence intensity of Gp31(I36W) is enhanced in the presence of
GroEL and nucleotide. Gp31(I36W) interacts with GroEL in the presence
of ATP as reported by an increase in fluorescence intensity as well as
a blue shift in max. Tryptophan was specifically excited at 295 nm,
and its emission was recorded over the range 320 to 380 nm.
,
Gp31(I36W);
, Gp31(I36W)-GroEL; ×, Gp31(I36W)·GroEL·ATP.
B, Gp31(I36W) was injected into a cuvette containing a
preequilibrated GroEL·ATP·cochaperonin complex. a,
GroEL + Gp31(I36W); b, GroEL·Gp31 + Gp31(I36W);
c, GroEL·Gp31(L35I,T31A) + Gp31(I36W); d,
GroEL·Gp31(L35I) + Gp31(I36W); e, Gp31(I36W).
Tryptophan was excited at 295 nm, and time-based emission was recorded
at 337 nm. In the absence of cochaperonin, Gp31(I36W) binds rapidly to
GroEL·ATP. The preaddition of Gp31 does not block binding but instead
retards binding of Gp31(I36W). The preaddition of Gp31(L35I) almost
completely inhibits binding of Gp31(I36W), whereas the preaddition of
Gp31(L35I,T31A) inhibits Gp31(I36W) binding to GroEL by ~50%.
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Fig. 4.
H /upfield region of NOESY
spectra of mobile loop peptides in the presence of GroEL. Spectra
were recorded at 30 °C as described under "Experimental
Procedures."
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Fig. 5.
X-ray and NMR structures. A,
a single subunit from the GroEL·ADP·GroES crystal structure (8).
The GroEL(E191G) and GroEL(A383T) specific amino acid changes are
localized in the hinges flanking the intermediate domain of GroEL and
are highlighted. B, the structure of the GroEL-bound GroES
mobile loop peptide was determined by trNOE NMR in the presence of
GroEL. The Ser21 and Ile25 residues (equivalent
to Thr31 and Leu35 of Gp31,
respectively) interact in the GroEL-bound conformation (24). Stuctures
were modeled using MolScript (44).
DISCUSSION
-strand preferences can account for the observed changes
in GroEL-Gp31 binding caused by the various mutations. A host-guest
study ranked all 20 amino acids by the change in free energy for
folding of a protein in which the substituted site was located at the
edge of a
-sheet (35). This study may be the most relevant because a
-hairpin more closely resembles the edge than the middle of a
-sheet. It was found that Leu is unfavorable for
-sheet
formation, Ala and Ile are neutral, whereas Thr is favorable. Thus, the
L35I substitution is expected to increase
-hairpin stability,
whereas either T31A or T31I are expected to decrease
-hairpin
stability. Because Gp31 binding to GroEL is coupled to
-hairpin
formation, changes in GroEL binding affinity may result from changes in
-hairpin stability. Minor and Kim (35) noted a significant
difference in the rank order of amino acids in
-sheet propensity for
substitution at the edge of a
-sheet versus the middle of
a
-sheet. In particular, Ile is a strong
-sheet-former in the
middle of a
-sheet but essentially neutral at its edge. The authors
suggested this is because of a balance of a favorable contribution from
side chain conformational entropy and poor hydrophobic burial at the
edge of the
-sheet. Thus, the initially surprising result that
either the T31A or T31I substitution can result in the same phenotype
can now be understood in terms of contributions to
-sheet stability.
-hairpin and yet
sufficiently disordered that GroEL binding is not too tight. As a
result, seemingly subtle amino acid substitutions, such as L35I, can
completely rescue or block bacteriophage T4 growth depending on the
particular mutant host.
-helix containing
Val264 twists when GroEL visits its open conformation. The
other two GroEL residues, Leu234 and Leu237,
show minor changes in orientation. Because mobile loop peptides exhibit
reduced binding to GroEL(E191G), mobile loop and, therefore, Gp31
binding may be controlled by the ratio of open versus closed GroEL subunits. If the mobile loop has a lower affinity for GroEL in
the closed conformation and GroEL(E191G) visits the open state less
frequently, then poor binding of Gp31 could be because of a smaller
population of GroEL(E191G) subunits in the open state.
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Fig. 6.
Location of GroEL residues directly bound by
the GroES mobile loop. Residues Leu234,
Leu237, and Val264 are presented as
space-filled atoms. A, a close-up view of the
apical domain in the GroEL crystal structure (3). B, a
close-up view of the GroEL apical domain with the GroES
mobile loop in the GroEL·ADP·GroES crystal structure (8).
Structures were modeled using MolScript (44).
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ACKNOWLEDGEMENTS |
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We thank Dr. Gisou van der Goot for her hospitality and help in using the luminescence spectrometer, Dr. Karol Maskos for help with NMR experiments, Dr. Alistair Kippen for help with the electrospray mass spectroscopy determinations, Françoise Schwager for assistance in DNA sequencing, Drs. Matthias Mayer, Jill Zeilstra-Ryalls, and Olivier Fayet for providing plasmids, Dr. Maciej Zylicz with help in protein overexpression, and Dr. David Boisvert for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Canton of Geneva, Swiss National Foundation Grant 31.47283-96 and National Science Foundation Grant MCB-9512711.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 07; Fax: 41-22 702 55 02; E-mail: Alexandra.Richardson{at}medecine.unige.ch.
¶ Present address: Faculty of Chemistry, Free University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
1 F. Keppel, unpublished data.
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ABBREVIATIONS |
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The abbreviation used is: trNOE, transferred nuclear Overhauser effect..
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REFERENCES |
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