Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036, India
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Abstract |
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Keywords: chaperonin-10/dome loop/metastable state/Mycobacterium tuberculosis/structural plasticity
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Introduction |
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Crystal structures of
E.coli
GroES (
Hunt et al., 1996
) as well as its homologue from
Mycobacterium leprae
(
Mande et al., 1996
) are known to atomic resolutions. GroES is a single heptameric ring of identical subunits. The overall structure of GroES resembles a dome with an orifice in its roof of ~812 Å. The GroES heptamer can cap either one or both ends of GroEL resulting in either an asymmetric (GroEL
14
):(GroES
7
) (
Xu et al., 1997
) or a symmetric (GroEL
14
):(GroES
7
)
2
complex (
Azem et al., 1994
).
The GroES monomer consists of a highly conserved hydrophobic core arranged in an irregular ß-barrel with a ß-hairpin extending from the top, named the `dome loop' (
Taneja and Mande, 1999
). Interestingly, the tip of the dome loop is dominated by a cluster of negatively charged residues. The tip of the dome loop is also the closest approach to the 7-fold symmetry axis in the chaperonin-10 (cpn-10) or GroES structures. The clustering of acidic residues, therefore, results in a very high negative potential towards the top of the GroES dome (
Hunt et al., 1996
;
Mande et al., 1996
). The concentration of acidic residues and the intense negative potential at the dome orifice may, hence, lead to an increased flexibility of the dome and possibly the instability of the oligomeric assembly. The GroES of
E.coli
has recently been proposed to have a metastable oligomeric structure leading to an increase in the diameter of the dome orifice in solution (
Timchenko et al., 2000
). In this study, we have attempted to characterize the flexibility of the dome loop by exploiting the presence of a single tryptophan residue in the dome loop of
Mycobacterium tuberculosis
cpn-10. Our results indicate that the negative charges at the dome are the likely factors responsible for the plasticity of cpn-10 structures.
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Materials and methods |
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Sequences of cpn-10 homologues were retrieved from the Swiss-Prot database (Release 36.0) (
Bairoch and Apweiler, 1999
). Sequence alignments were carried out using the ClustalW program (
Thompson et al., 1994
) employing the BLOSUM matrix (
Henikoff and Henikoff, 1993
). Default values of 10 and 0.05 for the gap opening and gap extension penalty, respectively, were used for the alignment.
Purification of cpn-10
Clones of
M.tuberculosis
cpn-10 in the pMAL-c expression vector (New England Biolabs) were available as a generous gift from Dr Vijay Mehra (Albert Einstein Institute of Medicine, New York, USA). Due to the choice of restriction sites in the multiple cloning sites, this construct consisted of seven additional residues incorporated at the N-terminus as observed from N-terminal sequencing of cpn-10 as well as from DNA sequencing of the gene. The additional residues were the same as reported in a similar cloning strategy for
M.tuberculosis
cpn-10 (
Rosenkrands et al., 1999
). These additional residues were deleted using the
Transformer
site-directed mutagenesis kit from Clontech. The deletion of the additional residues was confirmed by DNA sequencing.
The protein was purified according to the protocol previously described (
Mehra et al., 1992
). Briefly,
E.coli
JM109 containing the fusion construct (in pMAL-c) were grown to log-phase (
A
600
0.40.5
) and induced with 0.2 mM IPTG at 30°C overnight. The cells were then harvested by centrifugation. The pelleted bacteria were resuspended in 20 mM TrisHCl (pH 8.0), 200 mM NaCl, 5mM EDTA, 1 mM PMSF and lysed by sonication. The lysed cells were then centrifuged at 9000
g
to separate the cell debris. The fusion protein was purified from the crude extract by affinity chromatography over an amylose column. After elution from the column with 10 mM maltose, cpn-10 was cleaved from the maltose binding protein with 0.4% w/w factor Xa protease at room temperature for 12 h. The two proteins were finally separated over an anion exchange column (Mono Q, Pharmacia) using fast protein liquid chromatography. The purified protein appeared as a single band on 15% SDSPAGE (
Laemmli, 1970
) as detected by silver staining. The homogeneity of the protein was further confirmed over a Superdex-75 column (Pharmacia) by the elution of the protein as a single peak of 70 kDa. The concentration of the protein was determined by the method of Bradford (1976). This native cpn-10 was then pooled and dialyzed extensively against 10 mM TrisHCl (pH 7.5) and stored at 4°C for further use.
Fluorimetric measurements
Fluorimetric measurements were performed with a Perkin-Elmer LS50B luminescence spectrometer in a 1 ml capacity quartz cuvette at 25°C using an excitation wavelength of 295 nm. The emission spectrum was recorded between 310 and 390 nm without any correction for photomultiplier sensitivity. The excitation and emission slits were set at 5 and 7.5 nm, respectively.
The concentration of protein in each scan was kept at 20 µg ml 1 by diluting into 20 mM TrisHCl buffer (pH 7.5). Individual salt solutions were prepared in 20 mM TrisHCl (pH 7.5) and added to the protein solution to a final concentration of 0.5 or 10 mM. The spectra were recorded after an equilibration of at least 1 h at 25°C. The samples were centrifuged at 10 000 g for 10 min before recording the spectra with the clear supernatant. Appropriate buffer baselines were subtracted in each case. Each spectrum was an average of five scans.
To confirm that the shift in
max
of emission was due to the cations, each sample with 0.5 mM salt was incubated with the protein along with 20 mM EDTA for at least 1 h and the spectra were recorded again as above.
Unfolding of cpn-10 with guanidine hydrochloride
For recording the spectrum of completely unfolded cpn-10, the protein was incubated with 4 M guanidineHCl overnight at 25°C, both in the presence and absence of cations. The guanidine concentration was determined by measurement of refractive index as described by Pace et al . (1989). Appropriate buffer baselines were again subtracted in each case. Each spectrum was an average of five scans.
pH titration of GroES
cpn-10 was diluted into 50 mM acetate buffer (pH 4.2) to a final concentration of 20 µg ml 1 . The fluorescence spectra were then recorded upon excitation at 295 nm as described above to monitor the effect of pH on the fluorescence of tryptophan.
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Results |
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Figure
1A
shows the alignment of 58 cpn-10 sequences retrieved from the Swiss-Prot database. One of the conserved features in the sequence alignment is a ß-hairpin at the tip of the dome. The dome loop is formed by ~10 residues (4959 in
M.tuberculosis
sequence numbering
), with residues 5153 at the tip of the ß-hairpin. In almost all the sequences examined, at least one of the three residues in the ß-hairpin is acidic although position 51 is less conserved with a hydrophobic residue in many sequences. In the
M.tuberculosis
sequence, however, all the three residues in the ß-hairpin are acidic, namely aspartate-51, glutamate-52 and aspartate-53 (Figure 1B
).
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The roof ß-hairpin has earlier been postulated to be meta-stable (
Hunt et al., 1996
) and has recently been shown to have an increased dome orifice in solution (
Timchenko et al., 2000
). Introduction of a tryptophan (Trp) residue in the dome loop would help in monitoring the plasticity of the dome loop through changes in intrinsic Trp fluorescence. Among all the cpn-10 sequences retrieved from the Swiss-Prot database, only the mycobacterial sequences contain a Trp residue in the dome loop (Figure 1A
), making them ideal candidates for monitoring the plasticity of the loop. Therefore, we decided to study the plastic nature of the dome loop through the intrinsic fluorescence of this naturally occurring Trp residue in
M.tuberculosis
cpn-10.
Intrinsic Trp fluorescence of M.tuberculosis cpn-10
The fluorescence emission spectrum of the native protein due to the only Trp in its sequence shows a
max
of 352 nm. This suggests that the Trp is almost fully solvent exposed. The same protein when incubated with 4 M guanidineHCl results in a marginal decrease in fluorescence intensity and a fluorescence emission maximum at 355 nm (Figure 2
)
. There is, thus, a minor red shift in the emission maximum when the protein is unfolded.
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Effect of metal ions on fluorescence
In the presence of metal ions, fluorescence emission spectra showed dramatic results.
Table
I
shows the fluorescence emission maxima in the presence of an individual metal ion in each experiment. It is clear that monovalent cations have no effect on fluorescence. However, divalent cations exhibit significant shifts in the emission maximum towards lower wavelengths (Figure 2
). The shift in
max
in the presence of divalent cations suggests stabilization of the dome loop with a concomitant burial of the Trp into a different metastable state of cpn-10.
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Effect of pH titration on fluorescence
The fluorescence spectrum of the
M.tuberculosis
cpn-10 in 50 mM acetate buffer (pH 4.2) recorded in the absence of cations yields a fluorescence emission maximum at 341 nm. This amounts to a blue shift of ~11 nm as compared to the native protein in Tris buffer (pH 7.5) (Figure 2
). At pH 4.2, the dome carboxylates are expected to be protonated, losing their negative charge. The unionized states of the carboxylic groups are thus expected to result in a decrease in the negative potential at the top of the cpn-10. Consequently, there would be decreased repulsions between the ß-hairpins of the dome leading to the burial of the tryptophans in the hydrophobic interface.
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Discussion |
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We have attempted to study the conformational plasticity of cpn-10 by taking advantage of a single Trp residue in the dome loop of
M.tuberculosis
cpn-10. The Trp residue, which is present within the loop, behaves as if it is fully solvent exposed in the native structure. The emission maximum of its fluorescence shifts very little towards higher wavelength under fully denaturing conditions. There is, however, a dramatic shift in
max
of the Trp in the presence of divalent cations. The blue shift of ~811 nm in the presence of divalent cations is indicative of solvent shielding of the Trp as seen in the crystal structure of cpn-10. This result is further corroborated by restoration of
max
, as that in the native protein, in the presence of EDTA along with the divalent cations. The divalent cations therefore serve to stabilize the negative charges at the tip of the dome loop.
Additional evidence for the loss in flexibility is provided by blue shift in
max
of Trp at lower pH. Thus, when the acidic residues lose their charge, the environment of the Trp residue in the dome loop becomes less polar. The shift in Trp fluorescence in the presence of divalent cations, its restoration in the presence of EDTA, and a similar blue shift at acidic pH indicate that the plasticity of the dome loop is due to the negatively charged residues at its tip.
Escherichia coli
GroES has recently been shown to have an increased dome orifice in solution than seen in its crystal structure (
Timchenko et al., 2000
). While the orifice of the dome is only 8 Å wide in the crystal structure, it opens by as much as 1015 Å in solution. This may be a consequence of the intrinsic flexibility of the dome loop due to the negative charges as seen for
M.tuberculosis
GroES in this study. Although the significance of dome loop dynamics is as yet unclear, we believe that our demonstration of its flexibility should lead to a better understanding of its function. It has also been previously suggested that stoichiometric binding of Mg
2+
stabilizes the
E.coli
GroES protein (
Boudker et al., 1997
). A number of divalent cations have also been previously reported for the maintenance of the proper oligomeric state of
M.tuberculosis
GroES (
Fossati et al., 1995
). In this report, we have shown that the plasticity of cpn-10 is due to the high negative potential at the orifice of the dome and is modulated by various metal ions.
It is interesting to note that the chelation of magnesium ions by EDTA or CDTA (
trans
-1,2-diaminocyclohexane
N
,
N
,
N
',
N
'-tetraacetic acid
) disrupts the GroES:GroEL association (
Hayer-Hartl et al., 1995
;
Feltham and Gierasch, 2000
). One of the mechanisms for this disruption is the depletion of nucleotides from the GroEL pool. This results in the `open' conformation of GroEL as in the trans ring (
Xu et al., 1997
) or as in the stand-alone structure that cannot bind GroES. However, it is plausible that simultaneous conformational changes in GroES in the absence of divalent cations further reduce its affinity for GroEL. The dome loop might hence utilize the negative charges at its tip and their neutralization with metal ions to act as a `conformational switch' for interaction with GroEL.
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Notes |
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Acknowledgments |
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References |
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Received October 17, 2000; revised February 22, 2001; accepted March 2, 2001.