Ionic interactions at both inter-ring contact sites of

Ionic interactions at both inter-ring contact sites of
GroEL are involved in transmission of the allosteric
signal: A time-resolved infrared difference study
BEGON˜A SOT,1 FRITZTHOF VON GERMAR,2 WERNER MA¨NTELE,2
JOSE MARI´A VALPUESTA,3 STEFKA G. TANEVA,4,5 AND ARTURO MUGA1
1
Unidad de Biofı´ sica (CSIC-UPV/EHU) and Departamento de Bioquı´ mica y Biologı´ a Molecular,
Universidad del Paı´ s Vasco, 48080 Bilbao, Spain
2
Institut fu¨r Biophysik, Johan Wolfgang Goethe Universita¨t, D-60590 Frankfurt, Germany
3
Centro Nacional de Biotecnologı´ a (CSIC), Universidad Auto´noma de Madrid, 28049 Madrid, Spain
4
Institute of Biophysics, Bulgarian Academy of Sciences, Sofia, 1113, Bulgaria
(RECEIVED March 22, 2005; FINAL REVISION June 1, 2005; ACCEPTED June 13, 2005)
Abstract
The biological activity of the double-ring chaperonin GroEL is regulated by complex allosteric interactions, which include positive intra-ring and negative inter-ring cooperativity. To further characterize
inter-ring communication, the nucleotide-induced absorbance changes in the vibrational spectrum of the
chaperonin GroEL, of two single-point mutants suppressing one inter-ring ionic contact (E461K and
E434K) and of a single-ring version of this protein, were investigated by time-resolved infrared difference
spectroscopy. Interaction of the nucleotide with the proteins was triggered by its photochemical release
from a biologically inactive caged precursor [P3-1-(2-nitro) phenylethyl nucleotide]. The results indicate
that (1) ATP binding to the protein induces a conformational change that affects concomitantly both
intra-ring and inter-ring communication, and (2) the experimental absorbance changes are sensitive to
the double-ring structure of the protein. The characterization of the single-point, inter-ring mutants
demonstrates that ionic interactions at both contact sites are involved in the transmission of the allosteric
signal. However, both mutations have different effects on the inter-ring interface. While that of E461K
still retains ionic contacts sensitive to ATP binding, E434K shows spectroscopic features similar to those
of the single-ring version of the protein, therefore suggesting that electrostatic interactions at these
contact sites contribute differently to the stability of the inter-ring interface.
Keywords: GroEL; chaperonin; allosterism; infrared spectroscopy; protein conformation
The molecular chaperonin GroEL from Escherichia coli
is a member of the heat shock protein 60 (Hsp 60) class
of chaperones, composed of 14 identical subunits of 57.3
5
Present address: Unidad de Biofı´ sica (CSIC-UPV/EHU) and
Departamento de Bioquı´ mica y Biologı´ a Molecular, Universidad del
Paı´ s Vasco, 48080 Bilbao, Spain.
Reprint requests to: Arturo Muga, Unidad de Biofı´ sica (CSIC-UPV/
EHU) and Departamento de Bioquı´ mica y Biologı´ a Molecular, Universidad del Paı´ s Vasco, 48080 Bilbao, Spain; e-mail: [email protected];
fax: +34-94-6013500.
Abbreviations: EM, electron microscopy; TR-IR, time-resolved
infrared.
Article published online ahead of print. Article and publication date
are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051469605.
kDa assembled as two stacked, seven-membered rings
with a central cavity that provide a confined environment for protein folding (Braig et al. 1994). Each subunit
is composed of three domains: an equatorial domain
that contains the nucleotide-binding site and the interring contacts; an apical domain that binds nonfolded
proteins and the cochaperone GroES; and an intermediate domain that connects the apical and the equatorial
domains.
The functional cycle of the GroEL/GroES system
involves a sophisticated allosteric regulation by ATP.
There are two levels of allosterism as seen by steadystate kinetic studies of ATP binding and hydrolysis:
Protein Science (2005), 14:2267–2274. Published by Cold Spring Harbor Laboratory Press. Copyright ª 2005 The Protein Society
ps0514696
Sot et al.
Article RA
2267
Sot et al.
positive intra-ring and negative inter-ring cooperativity. The allosteric transitions have been characterized
by different biophysical techniques, such as fluorescence spectroscopy, electron microscopy, and smallangle X-ray scattering (Yifrach and Horovitz 1995;
Roseman et al. 1996; Llorca et al. 1997; Cliff et al.
1999; Ranson et al. 2001; Inobe et al. 2003). Nucleotide
binding and hydrolysis induce conformational changes
in the chaperonin, which cycles between protein substrate-binding and -release states (Staniforth et al. 1994;
Yifrach and Horovitz 1996, 2000). As a consequence of
the double-ring structure of GroEL, there are three
allosteric states—TT, TR, and RR—in which T (low
affinity for ATP and high affinity for nonfolded proteins) and R (high affinity for ATP and low affinity for
nonfolded proteins) represent the allosteric state of
each ring.
The importance of inter-ring allosteric communication in controlling the GroEL/GroES reaction cycle is
demonstrated by the fact that substrate and GroES
release from the GroES-bound cis ring depends on
ATP binding to the trans ring (Rye et al. 1997). Furthermore, the rate of GroES dissociation from the cis ring is
controlled by the cooperativity of ATP binding to the
trans ring—the stronger the intra-ring positive cooperativity, the faster the inter-ring communication. It follows
from these findings that the combination of inter- and
intra-ring cooperativity modulates the reaction cycle
time (Amir and Horovitz 2004).
Despite the key role of inter-ring allosteric signaling in
the biological function of GroEL, its molecular basis
remains poorly understood. As seen in the 3D structure
of the protein, the contact surface between the two rings
of the native protein involves both electrostatic and
hydrophobic interactions (Braig et al. 1994). Each subunit of a protein ring interacts with two of the opposite
rings through two contact sites. Among the most important interactions between opposite rings, the salt bridges
E461–R452 and E434–K105 are thought to stabilize the
inter-ring interface, which in turn allows proper interring communication at physiological temperature (Sot et
al. 2002, 2003; Sewell et al. 2004). The contact E434–
K105 is weaker in the ATP state (nucleotide-dependent),
these residues being at the end of an a-helix that crosses
the equatorial domain to the ATP-binding site. The
other salt bridge (E461–R452) appears in all the highresolution structures of apo and liganded GroEL (Boisvert at al. 1996; Roseman et al. 1996; Xu et al. 1997). We
have previously demonstrated that time-resolved infrared difference spectroscopy (TR-IR)1, in combination
with the use of caged nucleotides, is a suitable technique
to detect subtle conformational changes during nucleotide binding to GroEL and its subsequent hydrolysis
(von Germar et al. 1999). Here we analyze these
2268
Protein Science, vol. 14
conformational changes and compare the ATP-induced
structural transitions in wild-type GroEL, two singlepoint inter-ring mutants (E434K and E461K), and a
single-ring version of the protein (SR1). Our results
indicate that ionic interactions at both contact sites participate in inter-ring signaling.
Results
ATP-induced IR difference spectra
The experimental approach used here to detect the molecular events associated with nucleotide binding and its
subsequent hydrolysis, requires illumination of the sample containing the protein and the caged ligand with an
UV flash that releases the nucleotide and triggers the
ATPase reaction (McCray et al. 1980). Difference spectra are calculated from the spectrum measured before
the flash and the spectra recorded after nucleotide
release (Barth et al. 1991; Barth and Zscherp 2000).
Residues involved in ligand binding and/or hydrolysis
will contribute to the difference spectrum, while the
contribution of ‘‘passive’’ residues would be canceled
out. Besides protein and ligand bands, difference spectra
also contain signals from the photolysis reaction (von
Germar et al. 1999). Previous studies have established
that bands assigned to the photolytic release of nucleotides dominate the difference IR spectra below 1300
cm 1 (Barth et al. 1997). Above this frequency, i.e., in
the 1750–1300 cm 1 spectral region, the experimentally
detected absorbance changes contain contributions from
molecular events related to the photolysis reaction and
to protein conformational changes induced by nucleotide binding and/or hydrolysis.
ATP induces absorbance changes due to
intra- and inter-ring communication
Differential signals due to intra- and inter-ring communication could, in principle, be distinguished by comparing the IR difference spectra of wild-type GroEL and
SR1 in the presence of ATP. Prior to the description of
the ATP-induced IR difference spectra of these samples,
it should be mentioned that none of the mutants used
in this study are able to bind caged nucleotides (data
not shown), as previously shown for wild-type GroEL
(von Germar et al. 1999). This means that absorbance
changes due to both nucleotide binding and hydrolysis
can be experimentally observed.
The time course of the absorbance changes detected
upon nucleotide release to the medium, which under our
experimental conditions occurs within the first 33 msec
after the flash (Barth et al. 1997; von Germar et al.
1999), is shown in Figure 1. The photolysis reaction itself
Inter-ring communication in GroEL
Figure 1. Time dependence of the IR difference spectrum of GroEL (left panel) and SR1 (right panel) in the presence of ATP.
Measurements were performed after release of 0.8 mM ATP in samples containing 0.8 mM protein subunits, at 25 C in H2Obased buffer. Spectra were recorded at the indicated times after the photolysis flash. Average of 10 (GroEL) and 11 (SR1)
independent experiments. For the sake of comparison, the photolysis spectrum is also shown (bottom traces).
gives differential features at 1642 (+) and 1526 ( ) cm 1
(Fig. 1, bottom traces), and as shown previously (von
Germar et al. 1999), the IR difference spectrum of
GroEL recorded 60 msec after the flash displays positive
bands at 1652 and 1560 cm 1, small differential signals
at 1694 (+), 1688 ( )/1676 (+), and 1625 (+) cm 1,
and two negative absorbance changes at 1508 and 1546
cm 1 (Fig. 1, left panel). The amplitude of these absorbance changes increases during the first 2 sec and is
maintained for 5 sec, and at longer times the intensity
of the differential signals drops to a value similar to that
seen with ADP (von Germar et al. 1999; see below).
When the same experiment is performed in the presence
of SR1 instead of GroEL (Fig. 1, right panel), the following differences are observed within the first 2.8 sec
after nucleotide release: (1) modifications of the relative
intensities of differential signals located in the 1670–1694
cm 1 spectral region; (2) appearance of an absorbance
change at 1639 (+)/1626 ( ) cm 1 in the spectrum of
SR1; and (3) in contrast to what is observed for wildtype GroEL, the intensity of the absorbance change at
1560 cm 1 does not vary with time, while that at 1570
cm 1 increases for both proteins (Figs. 1, 2). After ATP
hydrolysis, the difference spectra of these samples look
alike (Fig. 1, top traces), and similar to those of the
ADP-bound conformation (von Germar et al. 1999; see
below).
The kinetic analysis of the absorbance changes at 1560
and 1570 cm 1, assigned to deprotonated side chains of
Glu and Asp residues, respectively (Chirgadze et al.
1975; Venyaminov and Kalnin 1990), indicates that the
ionization state of acidic amino acids of wild-type
GroEL and SR1 differs in the presence of ATP, whereas
it is similar in the ADP-bound state generated after
nucleotide hydrolysis (Fig. 2A). A similar time evolution
was observed for differential signals assigned to modifications of phosphate vibrations (1268 cm 1) and of the
amide I mode of the protein backbone (1652 cm 1)
(Barth and Zscherp 2002), as previously suggested (von
Germar et al. 1999) (Fig. 2B). To assign the observed
kinetic events, experiments were performed at different
ATP/GroEL subunit molar ratios (Fig. 3). When the
molar ratio is 1, the initial exponential intensity increase
that occurs for wild-type GroEL at 3–4 sec 1 is followed by a lag phase before a single exponential intensity decay takes place at 0.07–0.09 sec 1 (Fig. 3A).
Under the same experimental conditions, the lag phase
is not observed for SR1, which shows the other two
kinetic processes with time constants of 4–6 sec 1 and
0.06–0.08 sec 1, respectively (Fig. 3A). In contrast, when
the ATP/wild-type GroEL subunit molar ratio is 0.5, the
lag phase is not detected, and increasing the molar ratio
to 2 results in the disappearance of the exponential decay
during the first 40 sec after ATP release to the medium
(Fig. 3B). For comparison, previously reported changes
in fluorescence intensity of pyrenyl-GroEL in the presence of ATP are shown as solid lines (Kad et al. 1998).
The initial fluorescence enhancement (k = 2 sec 1) was
assigned to the conformational rearrangement of the
occupied ring to the R-state, whereas the exponential
www.proteinscience.org
2269
Sot et al.
Figure 2. Kinetics of selected infrared absorbance changes of GroEL
and SR1 induced by ATP. (A) Time dependence of the absorbance
changes at 1570 (circles) and 1560 (squares) cm 1 for GroEL (filled
symbols) and SR1 (open symbols). (B) Time dependence of the intensity of differential signals at 1655 (squares) and 1268 cm 1 (triangles).
Different constants were added to the values obtained for GroEL
(filled symbols) and SR1 (open symbols) to allow for a simultaneous
representation. Spectra were recorded in H2O-based buffer as described in Figure 1.
interface, since mutations affecting these ionic interactions have a clear effect on the inter-ring spacing, the
folding activity, and the thermal stability of the oligomeric particle (Sot et al. 2002, 2003). To follow in realtime the evolution of the ATP-induced absorbance
changes that could be assigned to inter-ring ionic interactions, we have generated single-point GroEL mutants
in which one of the salt bridges was suppressed (E434K
and E461K). The spectroscopic characterization of these
mutants should help to decipher whether ionic interactions at these contact sites are involved in the transmission of the allosteric signal that promotes GroES and
protein substrate release from one protein heptamer,
upon ATP binding to the opposite ring. Data obtained
in D2O buffer for the four proteins are presented in
Figure 4, together with the spectra obtained after subtracting the contribution from the photolysis reaction
(inset). The comparison with results obtained in H2O
medium allows to distinguish changes due to amide
modes of the polypeptide backbone from those caused
by amino acid side chains (see Figs. 1, 4). Deuteration
shifts the amide I modes (1700–1610 cm 1), mainly due
to peptide C=O groups, up to 10 cm 1, and the amide II
band, mainly due to amide-backbone NH groups, from
1550 cm 1 to 1460 cm 1 (Barth and Zscherp 2002). In con-
decrease in fluorescence intensity occurs at 0.1 sec 1,
the rate of ATP hydrolysis (Kad et al. 1998). The good
agreement between the rate constants determined by
Kad et al. (1998) and the values estimated in the present
work allows us to assign the first kinetic event to the Tto-R conformational transition, the lag phase to the
steady-state hydrolysis of ATP, where the nucleotide is
continuously binding to the protein, and the decay phase
to nucleotide hydrolysis.
Electrostatic interactions at both contact sites
contribute to inter-ring signaling
Assuming that the ATP-induced allosteric intra-ring
conformational change is the same for the double- and
single-ring version of GroEL, the data presented so far
suggest that (an) acidic side chain(s) might be involved in
inter-ring communication. As pointed out above, and in
previous studies (Braig et al. 1994; Sot et al. 2002, 2003;
Sewell et al. 2004), salt bridges could be potentially
established at both contact sites of the inter-ring
2270
Protein Science, vol. 14
Figure 3. (A) Time dependence of the absorbance changes at 1655
(circles) and 1568 (squares) cm 1 for GroEL (filled symbols) and SR1
(open symbols). The nucleotide/protein subunit molar ratio was 1 in
both cases. (B) Kinetics of the 1568 cm 1 differential signal obtained at
nucleotide/wild-type GroEL subunit molar ratios of 0.5 (filled squares)
and 2 (open circles). Other details are as in Figure 2. The fluorescence
changes of pyrenyl-GroEL in the presence of ATP (nucleotide/GroEL
subunit molar ratio 1 [A] and 0.5 [B]) are shown as solid lines (Kad
et al. 1998).
Inter-ring communication in GroEL
Figure 4. ATP-induced IR absorbance changes in D2O medium. The
difference spectra were recorded 3 sec after release of 1.6 mM ATP in
samples containing 0.8 mM protein subunits. Wild-type GroEL (thick
solid line), E461K (dashed/dotted line), E434K (dotted line), and SR1
(thin solid line). Photolysis reaction recorded under identical experimental conditions but in the absence of proteins (). Data are the
average of 15 (GroEL), 10 (E461K), 12 (E434K), and 16 (SR1) different experiments. Other details are as in Figure 1. Due to the higher
intrinsic ATPase activity of mutants E461K and E434K (Sot et al.
2002), the amount of ATP released in these samples was 1.6 mM to
extend the binding event to 3 sec. (Inset) Difference spectra obtained
after subtracting the photolysis spectrum from those obtained in the
presence of the proteins. The criterion used to choose the subtraction
factor was to cancel the negative signal at 1526 cm 1, indicated by an
arrow.
trast, larger downshifts ( 30 cm 1) are characteristic for
the side-chain absorptions of solvent-accessible Asn,
Gln, Lys, and Arg, as are small upshifts of up to 10
cm 1 for COO bands (Chirgadze et al. 1975; Venyaminov and Kalnin 1990; Barth 2000).
As compared with the spectra recorded in H2O, the IR
difference spectrum of GroEL measured 2 sec after ATP
release in D2O buffer shows the following differences
(Fig. 4): (1) The differential feature described in H2O
at 1694 cm 1 disappears; (2) the absorbance change at
1652 cm 1 is downshifted to 1648 cm 1; (3) the absor-
bance changes at 1546 ( ) and 1508 ( ) cm 1 are significantly weaker and disappear, respectively; and (4) the
positive band at 1560 cm 1 is slightly upshifted upon
deuteration. Differences at 1625 cm 1 are maintained in
D2O. These findings reinforce the assignment of 1651
(H2O) and 1647 cm 1 (D2O) to helical segments of the
protein (Byler and Susi 1986), in accordance with the
helical structure of the equatorial domain (Braig et al.
1994); the 1546 ( ) cm 1 differential signal to amide II
modes of the protein; and the 1508 ( ) cm 1 absorbance
change to NH3 modes of Lys (von Germar et al. 1999).
As compared with data obtained with wild-type GroEL,
there are several differences in the IR spectrum of the
ATP-bound state of SR1, namely, (1) the positive signal
at 1627 cm 1 disappears in the spectrum of SR1, and
instead a negative differential feature is observed at 1631
cm 1, as described in H2O; and (2) the intensity of the
absorbance changes previously assigned to acidic amino
acids at 1584 and 1567 cm 1 decreases (Fig. 4). Interestingly, the amplitude of these differential features
observed in the spectrum of E461K is intermediate
between those seen for wild-type GroEL and SR1,
whereas the difference spectrum of E434K resembles
that of the single-ring version of the protein (Fig. 4). It
is important to note that these differences are only
detected when comparing the ATP-bound states of the
proteins, e.g., during the first 2 sec after nucleotide
release to the medium, and that they are abolished
once ATP is completely hydrolyzed, as already mentioned in H2O buffer. Since under our experimental
conditions both single-point mutants share a doublering structure (Sot et al. 2002), these data indicate that
the mutated residues participate in inter-ring signaling.
ADP-induced conformational changes
The absorbance changes induced upon ADP binding to
these proteins are shown in Figure 5, together with those
detected during the photolysis reaction. At variance with
ATP, ADP binding does not induce any significant
spectral difference between these proteins, and therefore
promotes a similar conformational rearrangement that
does not involve the inter-ring interface. As expected,
none of these differential bands show the kinetic behavior described in the presence of ATP.
Discussion
GroEL is believed to function as a two-stroke complex,
in which both rings participate in a negative allosteric
mechanism that synchronizes binding and hydrolysis of
ATP with binding and release of GroES and protein
substrates (Lorimer 1997). Although each of the two
rings is the functional unit in protein folding (Weissman
www.proteinscience.org
2271
Sot et al.
Figure 5. ADP-induced absorbance changes in D2O medium. Difference spectra recorded 3 sec after the release of 0.8 mM ADP into
samples containing 0.8 mM protein subunits. Wild-type GroEL (thick
solid line), E461K (dashed/dotted line), E434K (dotted line), and SR1
(thin solid line). Photolysis spectrum (). Average of seven (GroEL),
eight (E461K), five (E434K), and eight (SR1) experiments.
et al. 1995), the double-ring structure and the signaling
between the two rings are stringent for the folding
mechanism to work (Rye et al. 1997). In this context,
the ATPase cycle of wild-type GroEL, two single-point
inter-ring mutants, and a single-ring version of the protein, has been followed by difference IR spectroscopy to
further understand the structural requirements that
allow transmission of the inter-ring allosteric signal.
The recently described consequences of the single
replacements (E461K and E434K) include a downshift
of the inactivation temperature (Sot et al. 2002) and a
decreased stability of the tetradecameric protein particle
against the thermal challenge (Sot et al. 2003). These
observations were interpreted as a result of a destabilization of the inter-ring interface, which causes a loss of
inter-ring negative cooperativity and favors dissociation
into heptamers with increasing temperatures. The conclusion of these studies was that ionic interactions at
both contact sites are required to maintain the functionality and stability of the inter-ring interface that allows
GroEL to function as a molecular thermometer distin2272
Protein Science, vol. 14
guishing physiological from stress temperatures (Sot
et al. 2002, 2003). A recent cryoEM study of one of the
mutants used in this study (E461K) proposes that a new
set of electrostatic contacts might be established at the
inter-ring interface, as a consequence of a reorganization
of this protein region that undergoes an 18 rigid body
rotation about the sevenfold symmetry axis of one of the
rings relative to the other (Sewell et al. 2004). As compared with wild-type GroEL, the contacts between subunits of opposing rings would be in E461K 1:1 instead of
1:2. Therefore, the electrostatic interactions left at the
inter-ring interface of the mutant would be E434–K470
and E102–K105, whereas in wild-type GroEL are E434–
K105 and E461–R452 (Braig et al. 1994; Xu et al. 1997).
The results presented here support the view that upon
ATP binding to GroEL, the initially observed protein
conformational transition most likely represents the rearrangement of the occupied ring from the T-state to the Rstate, while nucleotide hydrolysis relaxes the protein to an
ADP-bound-like conformation. An important finding in
this study is that this structural transition, as seen by IR
spectroscopy, is sensitive to the double-ring structure of
the protein and, therefore, witnesses inter-ring signaling.
Although the simultaneous presence of other differential
features in the IR spectra of these proteins also reveals an
ATP-induced intra-ring conformational rearrangement,
we focus on inter-ring communication. However, it is
interesting to point out here that the time evolution of
the absorbance changes that might be assigned to interand intra-ring conformational events is similar, indicating, as previously suggested (Shiseki et al. 2001; Amir and
Horovitz 2004), that they are both coupled.
ATP binding to GroEL has been suggested to distort the
inter-ring interface, inducing an overall increase in separation of the rings and tilts of the equatorial domains of the
protein (Ranson et al. 2001). Analysis of the absorbance
changes appearing only in the difference spectra of wild-type
GroEL and not in that of SR1, e.g., those involved in interring communication, clearly demonstrates that during the
T-to-R conformational transition, the chemical environment of an acidic, most likely a Glu, residue(s)s in GroEL
(Figs. 2, 3) is altered. This is not caused by the ionization of –
COOH group(s) that would contribute with a negative
signal at 1700–1730 cm 1, and might reflect the rearrangement of the inter-ring interface that causes modification of
ionic interactions between residues located in subunits of
opposite rings. The assignment of an arginine residue as the
counterion of E461 is suggested by (1) the absorbance
changes observed in H2O at 1680 (+)/1670 ( ) and 1642
( )/1635 (+) cm 1 and (2) the decreased intensity of these
differential signals upon deuteration (see Figs. 1, 3). A
differential signal at similar frequencies has been assigned
to an Arg residue that was involved in ion-pairing interaction with a halide ion in halorhodopsin (Braiman et al. 1994;
Inter-ring communication in GroEL
Rudiger et al. 1995), and reinforces the idea that the ion
pair E461K–R452 senses ATP binding to GroEL. It is
also clear from our data that at the inter-ring interface of
E461K there are still ionic interactions sensitive to ATPinduced inter-ring communication, that might include
those left at the other contact site or a new set of charged
contacts, as recently proposed (Sewell et al. 2004). It
should also be mentioned that this assignment is based
on the comparison of nucleotide-induced difference spectra of wild-type and single-point, double-ring protein
structures and SR1. Therefore, we cannot rule out that
other ionic contacts might also contribute to the differential signals during the T-to-R transition, or that these
mutations might also have some effects on these interactions (White et al. 1997).
A different picture is obtained with E434K GroEL.
Disruption of the ionic contacts at this site results in the
disappearance of the absorbance changes assigned to
inter-ring communication in the TR state of wild-type
GroEL. Instead, the difference spectrum of this mutant
resembles that of SR1 in spite of showing a double-ring
structure under our experimental conditions. The interring interface of E434K senses the T-to-R transition
differently, as compared with E461K, a behavior that
might be caused by (1) a new interaction between subunits of opposing rings that place oppositely charged
residues far enough apart to cancel out Coulombic
attractions under the experimental conditions used in
this study (e.g., 50 mM NaCl, 30 mM MgCl2), and/or
(2) a destabilization of the inter-ring interface that
increases inter-ring distance (Sot et al. 2002; Sewell et
al. 2004) and hampers interaction between residues that
might form salt bridges in wild-type GroEL. In this
context, it is important to note that this interpretation
would be in excellent agreement with the stronger effect
of mutation E434K in destabilizing the inter-ring interface (Sot et al. 2003), which might result in a larger interring spacing and therefore weaker electrostatic interactions, as experimentally observed.
In summary, the present work demonstrates that upon
ATP binding, the T-to-R transition involves a rearrangement of the inter-ring interface of GroEL that affects the
ionic interactions at both contact sites. It also shows that
these interactions are required to efficiently transmit the
inter-ring allosteric signal, essential to maintain the
asymmetric reaction cycle and thus the functionality of
the chaperonin.
Materials and methods
Protein expression and purification
GroEL mutants were produced by the homologous recombination technique (Martin et al. 1995), according to Weissman
et al. (1995). Wild-type GroEL and mutants were overexpressed from E. coli, purified as described previously (Sot et
al. 2002), and concentrated using microconcentration filters
(Centricon 50; Amicon).
Sample preparation
Sample buffer was exchanged by repeated concentration and
dilution steps with a buffer of 100 mM MOPS, 50 mM NaCl,
and 30 mM MgCl2 made in H2O or D2O (pH or pD 7.0). IR
samples were prepared by drying onto a CaF2 window 1 mL of
the following reagents: caged nucleotide (5, 10, or 40 mM), 40
mM KCl, and 20 mM DTT. They were rehydrated with the
same volume of the desired protein dissolved in the above
buffer, and the samples were sealed with a second window.
The protein subunit concentration was 0.8 mM, as estimated
with the bicinchoninic acid assay (Sigma). All experiments
were carried out at 25 C, using thermostated cell holders.
Time-resolved infrared spectroscopy (TR-IR)
Experiments were performed in modified IFS 66 (Bruker)
and Nexus 870 (Thermo) spectrophotometers equipped with
MCT detectors. Photolytic release of nucleotides from their
caged derivatives was triggered with a Xenon flash tube. The
voltage of the flash power supply was adjusted to release the
desired nucleotide concentration. Data were acquired with
double-sided interferograms, at a spectral resolution of 4 and
8 cm 1.
To improve the signal-to-noise ratio, signals obtained from
several samples were averaged after normalizing the spectra to
an identical protein concentration. Normalization prevents the
possible predominance of individual samples with the highest
protein concentration in the averaged spectra, and was done as
previously described (von Germar et al. 2000). Control experiments on samples prepared as described above but without
protein were performed in the same time interval to obtain
the photolysis spectra. They were subtracted from the protein
spectra to get bands that mainly arise from nucleotide binding
and its subsequent hydrolysis, as described. The criterion used
to choose the subtraction factor was to cancel the negative
signal at 1526 cm 1 (von Germar et al. 2000). For the kinetic
analysis of ATP-induced difference spectra, selected bands
were integrated as described previously (Barth et al. 1991;
von Germar et al. 2000). Briefly, the time slots of spectra
recording were represented by their average times, and the
time constants were obtained by fitting band intensities to
single exponential functions (Origin 5.0).
Miscellaneous methods
A spectrophotometric method that includes an ATP regenerating system was used to characterize the ATPase activity of the
proteins (Sot et al. 2002). Nucleotide binding was assayed by
mixing 150 mM free and caged nucleotides with the same
concentration of protein subunits. The mixture was centrifuged
in a Microcon-30 (Amicon) microconcentration filter, and the
concentration of the unbound nucleotide that passed through
the filtration membrane was estimated using an extinction
coefficient of 15,400 M 1 cm 1. Control experiments without
nucleotide were performed to subtract background contributions to this value.
www.proteinscience.org
2273
Sot et al.
Acknowledgments
B.S. and S.G.T. were recipients of fellowships from the Basque
Government and the University of the Basque Country. We
thank F.M. Gon˜i and F. Moro for critically reading the manuscript. A. Galan and O. Llorca are gratefully acknowledged for
preliminary results. This work was supported by grants from
the MEC (BFU2004-03452/BMC) and the University of the
Basque Country (13505/2001).
References
Amir, A. and Horovitz, A. 2004. Kinetic analysis of ATP-dependent interring communication in GroEL. J. Mol. Biol. 338: 979–988.
Barth, A. 2000. The infrared absorption of amino acid side chains. Prog.
Biophys. Mol. Biol. 74: 141–173.
Barth, A. and Zscherp, C. 2000. Substrate binding and enzyme function
investigated by infrared spectroscopy. FEBS Lett. 477: 151–156.
———. 2002. What vibrations tell us about proteins. Q. Rev. Biophys. 35:
369–430.
Barth, A., Ma¨ntele, W., and Kreutz, W. 1991. Infrared spectroscopic signals
arising from ligand binding and conformational changes in the catalytic
cycle of sarcoplasmic reticulum calcium ATPase. Biochim. Biophys. Acta
1057: 115–123.
Barth, A., Corrie, J.E.T., Gradwell, M.J., Maeda, Y., Ma¨ntele, W., Meier,
T., and Trentham, D.R. 1997. Time-resolved infrared spectroscopy of
intermediates and products from photolysis of 1-(2-nitrophenyl)ethyl
phosphates: Reaction of the 2-nitrosoacetophenone byproducts with
thiols. J. Am. Chem. Soc. 119: 4149–4159.
Boisvert, D.C., Wang, J.M., Otwinowski, Z., Horwich, A.L., and Sigler,
P.B. 1996. The 2.4 A˚ crystal-structure of the bacterial chaperonin GroEl
complexed with ATP-gS. Nat. Struct. Biol. 3: 170–177.
Braig, K., Otwinowski, R., Edge, D.C., Boisvert, A., Joachimiak, A.,
Horwich, A.L., and Sigler, P.B. 1994. The crystal structure of the
bacterial chaperonin GroEL at 2.8 A˚ resolution. Nature 371: 578–586.
Braiman, M.S., Walter, T.J., and Briercheck, D.M. 1994. Infrared spectroscopic detection of light-induced change in chloride-arginine interaction in halorhodopsin. Biochemistry 33: 1629–1635.
Byler, D.M. and Susi, H. 1986. Examination of the secondary structure of
proteins by deconvolved FTIR spectra. Biopolymers 25: 469–487.
Chirgadze, Y.N., Fedorov, O.V., and Trushina, N.P. 1975. Estimation of
the amino acid residue side-chain absorption in the infrared spectra of
protein solutions in heavy water. Biopolymers 14: 679–694.
Cliff, M.J., Kad, N.M., Hay, N., Lund, P.A., Webb, M.R., Burston, S.G.,
and Clarke, A.R. 1999. A kinetic analysis of the nucleotide-induced
allosteric transitions of GroEL. J. Mol. Biol. 293: 667–684.
Inobe, T., Arai, M., Nakao, M., Ito, K., Kamagata, K., Makio, T.,
Amemiya, Y., Kihara, H., and Kuwajima, K. 2003. Equilibrium and
kinetics of the allosteric transition of GroEL studied by solution X-ray
scattering and fluorescence spectroscopy. J. Mol. Biol. 327: 183–191.
Kad, N.M., Ranson, N.A., Cliff, M.J., and Clarke, A.R. 1998. Asymmetry,
commitment and inhibition in the GroEL ATPase cycle impose alternating functions on the two GroEL rings. J. Mol. Biol. 278: 267–278.
Llorca, O., Marco, S., Carrascosa, J.L., and Valpuesta, J.M. 1997. Conformational changes in the GroEL oligomer during its functional cycle.
J. Struct. Biol. 118: 31–42.
Lorimer, G.H. 1997. Protein folding. Folding with a two-stroke motor.
Nature 388: 720–721.
Martin, A., Toselli, E., Rosier, M., Auffray, C., and Devignes, M. 1995. Rapid
and high efficiency site-directed mutagenesis by improvement of the homologous recombination technique. Nucleic Acids Res. 23: 1642–1643.
2274
Protein Science, vol. 14
McCray, J.A., Herbette, L., Kihara, T., and Trentham, D.R. 1980. A new
approach to time-resolved studies of ATP-requiring biological systems:
Laser flash photolysis of caged ATP. Proc. Natl. Acad. Sci. 77: 7237–
7241.
Ranson, N.A., Farr, G.W., Roseman, A.M., Gowen, B., Fenton, W.A.,
Horwich, A.L., and Saibil, H.R. 2001. ATP-bound states of GroEL
captured by cryo-electron microscopy. Cell 107: 869–879.
Roseman, A.M., Chen, S., White, H., Braig, K., and Saibil, H.R. 1996. The
chaperonin ATPase cycle: Mechanism of allosteric switching and
movements of substrate-binding domains in GroEL. Cell 87: 241–251.
Rudiger, M., Haupts, V., Gerwert, K., and Oesterhelt, D. 1995. Chemical
reconstitution of a chloride pump inactivated by a single point mutation. EMBO J. 14: 1599–1606.
Rye, H.S., Burston, S.G., Fenton, W.A., Beechem, J.M., Xu, Z., Sigler,
P.B., and Horwich, A.L. 1997. Distinct actions of cis and trans ATP
within the double ring of the chaperonin GroEL. Nature 388: 792–
798.
Sewell, B.T., Best, R.B., Chen, S., Roseman, A.M., Farr, G.W., Horwich,
A.L., and Saibil, H.R. 2004. A mutant chaperonin with rearranged
inter-ring electrostatic contacts and temperature-sensitive dissociation.
Nat. Struct. Mol. Biol. 11: 1128–1133.
Shiseki, K., Murai, N., Motojima, F., Hisabori, T., Yoshida, M., and
Taguchi, H. 2001. Synchronized domain-opening motion of GroEL is
essential for communication between the two rings. J. Biol. Chem. 276:
11335–11338.
Sot, B., Galan, A., Valpuesta, J.M., Bertrand, S., and Muga, A. 2002. Salt
bridges at the inter-ring interface regulate the thermostat of GroEL.
J. Biol. Chem. 277: 34024–34029.
Sot, B., Ban˜uelos, S., Valpuesta, J.M., and Muga, A. 2003. GroEl stability
and function. Contribution of the ionic interactions at the inter-ring
contact sites. J. Biol. Chem. 278: 32083–32090.
Staniforth, R.A., Burston, S.G., Atkinson, T., and Clarke, A.R. 1994.
Affinity of chaperonin-60 for a protein substrate and its modulation
by nucleotides and chaperonin-10. Biochem. J. 300: 651–658.
Venyaminov, S.Y. and Kalnin, N.N. 1990. Quantitative IR spectrophotometry of peptide compounds in water (H2O) solutions. II. Amide
absorption bands of polypeptides and fibrous proteins in a-, b-, and
random coil conformations. Biopolymers 30: 1259–1271.
von Germar, F., Gala´n, A., Llorca, O., Carrascosa, J.L., Valpuesta, J.M.,
Ma¨ntele, W., and Muga, A. 1999. Conformational changes generated
in GroEL during ATP hydrolysis as seen by time-resolved infrared
spectroscopy. J. Biol. Chem. 274: 5508–5513.
von Germar, F., Barth, A., and Ma¨ntele, W. 2000. Structural changes of
the sarcoplasmic reticulum Ca2+-ATPase upon nucleotide binding
studied by Fourier transform infrared spectroscopy. Biophys. J. 78:
1531–1540.
Weissman, J.S., Hohl, C.M., Kovalenko, O., Kashi, W.A., Chen, S., Braig,
K., Saibil, H.R., Fenton, W.A., and Horwich, A.L. 1995. Mechanism
of GroEL action: Productive release of polypeptide from a sequestered
position under GroES. Cell 83: 577–588.
White, H.E., Shaoxia, C., Roseman, A.M., Yifrach, O., Horovitz, A., and
Saibil, H.R. 1997. Structural basis of allosteric changes in the GroEL
mutant Arg197-Ala. Nat. Struct. Biol. 4: 690–694.
Xu, Z., Horwich, A.L., and Sigler, P.B. 1997. The crystal structure of the
asymmetric GroEL–GroES–(ADP)7 chaperonin complex. Nature 388:
741–750.
Yifrach, O. and Horovitz, A. 1995. Nested cooperativity in the ATPase
activity of the oligomeric chaperonin GroEL. Biochemistry 34: 5303–
5308.
———. 1996. Allosteric control by ATP of non-folded protein binding to
GroEL. J. Mol. Biol. 255: 356–361.
———. 2000. Coupling between protein folding and allostery in the GroE
chaperonin system. Proc. Natl. Acad. Sci. 97: 1521–1524.

Download Report