From the Departments of Biophysical Chemistry and
¶ Structural Biology, Biozentrum, University of Basel,
Klingelbergstrasse 70, CH-4056 Basel, Switzerland and the
Oncology Research and ** Functional Genomics Area,
Novartis Pharma AG, CH-4002 Basel, Switzerland
Received for publication, November 20, 2000, and in revised form, December 26, 2000
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ABSTRACT |
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We previously reported that a helical trigger
segment within the GCN4 leucine zipper monomer is indispensable for the
formation of its parallel two-stranded coiled coil. Here, we
demonstrate that the intrinsic secondary structure of the trigger site
is largely stabilized by an intrahelical salt bridge. Removal of this
surface salt bridge by a single amino acid mutation induced only minor
changes in the backbone structure of the GCN4 leucine zipper dimer as
verified by nuclear magnetic resonance. The mutation, however,
substantially destabilized the dimeric structure. These findings
support the proposed hierarchic folding mechanism of the GCN4 coiled
coil in which local helix formation within the trigger segment precedes dimerization.
The role of intrinsic secondary structure in promoting structure
formation is one of the central questions in protein folding and a
major subject of debate. Because of its simplicity and regularity, the
coiled-coil structural motif has frequently been used as a model system
for experimental studies of protein folding. One of the simplest and
most widely characterized coiled coils is the parallel two-stranded
33-residue leucine zipper from the yeast transcriptional activator GCN4
(1). We recently postulated that a segment of the GCN4 leucine zipper,
termed the "trigger sequence," drives coiled-coil formation by
stabilizing an Plasmid Constructions, Protein Expression, and Peptide
Synthesis--
Synthetic genes encoding the wild-type and mutant
proteins were prepared with optimal codon usage for Escherichia
coli (17) and ligated into the BamHI/EcoRI
site of pPEP-T (2).
E. coli JM109(DE3) host strain (Promega) was used for
expression. Production and purification of 6xHis-tagged fusion proteins by affinity chromatography on Ni2+-Sepharose (Novagen) and
separation of recombinant GCN4 proteins from the carrier protein were
performed as described in the manufacturer's instructions. Recombinant
proteins were dialyzed against 10 mM sodium phosphate
buffer (pH 7.0) containing 150 mM sodium chloride.
Peptides were assembled on an automated continuous flow synthesizer
employing standard methodologies (18). The purity of the peptides was
verified by reversed-phase analytical high pressure liquid
chromatography, and the identity of the final products was
verified by amino acid and mass spectral analyses. Protein and peptide
concentrations were determined by tyrosine absorbance at 280 nm in 6 M GuHCl (19).
Biophysical Characterization--
Nuclear magnetic resonance
(NMR) experiments were acquired on a 600-MHz Bruker DRX spectrometer.
NMR assignments for GCN4p-wt and GCN4p-R25A were based on
three-dimensional NOESY1-HSQC
(mixing time,
Analytical ultracentrifugation and CD spectroscopy were
performed as described previously (2, 5).
The secondary structures, thermal stabilities, and subunit
stoichiometries of the recombinant GCN4 polypeptide chain fragments (Fig. 1A) were analyzed by CD
spectroscopy (Fig. 1, B and C) and analytical
ultracentrifugation (Table I).
Consistent with the previously reported observations (2, 22), the
wild-type GCN4 leucine zipper (GCN4p-wt) formed a dimer with a CD
spectrum typical of
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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-helical segment within the polypeptide monomer (2).
This mechanism has been confirmed by two independent research groups
(3, 4). Trigger sequences are conserved in many coiled coils and are
required for dimerization of the GCN4 leucine zipper (2), the
actin-bundling protein cortexillin I from Dictyostelium
discoideum (5, 6), for specific trimer formation of the macrophage
scavenger receptor (7), and the stability of keratin intermediate
filaments (8). We demonstrated that trigger sequences represent
autonomous helical folding units, which differ from arbitrarily chosen
heptad repeats by their ability to mediate coiled-coil formation (2,
5). Because favorable side chain interactions are critical for monomer helix stability of the isolated GCN4 trigger sequence (2), the
intrahelical surface salt bridge between Glu22 and
Arg25 seen in crystal structures of GCN4 (1, 9, 10)
potentially could affect the stability of the leucine zipper dimer. To
test this hypothesis, we designed a GCN4 leucine zipper mutant,
designated GCN4p-R25A, in which this salt bridge is missing. As
controls, we generated two additional variants in which the other
observed salt bridge between Lys8 and Glu11
outside of the trigger sequence (GCN4p-E11A) or both ionic interactions (GCN4p-E11A/R25A) were removed. For the purposes of this study, only
Glu11 or/and Arg25 was selected for
substitution because these residues at heptad c positions
are not thought to be essential for maintaining the coiled-coil
structure (11, 12). In contrast, Lys8 and Glu22
at heptad g positions were fixed because these residues
contribute to the hydrophobic core of the leucine zipper (1). To
exclude the possibility of a helix destabilizing effect due to a
lower helix propensity of the mutant residue, alanine was used for
substitution. Alanine was reported to have the highest helix propensity
in several peptides (13, 14), at the f position in coiled
coils (11) and at exposed helical sites in proteins (15, 16). The
mutant proteins investigated in this study are shown in Fig.
1A.
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ABSTRACT
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RESULTS AND DISCUSSION
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= 100 ms), TOCSY-HSQC (
= 60 ms),
HMQC-NOESY-HMQC (
= 150 ms ), and HNHA experiments (20, 21).
The experiments were recorded on 2.4 mM samples of
GCN4p-R25A, and 1.4 mM samples of GCN4p-wt (monomer
concentrations) in 90% H2O, 10% D2O
containing 150 mM sodium chloride, pH 6.2 ± 0.2, at a
temperature of 35 °C. 3JHNH
coupling
constants were calculated from the ratio of diagonal to cross-peak
intensities in three-dimensional HNHA spectra using the approach
described by Vuister and Bax (20). Hydrogen-deuterium exchange in the
GCN4p-R25A mutant was monitored using a 1.4 mM solution
(monomer concentration), freshly dissolved in D2O
containing 150 mM NaCl, at a pD of 6.1 and a temperature of
5 °C.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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-helix structure and with a sigmoidal thermal
unfolding profile. GCN4p-E11A also folded into a dimer with an
-helix content and a thermal stability comparable with those of the
wild-type protein. In contrast, substitution of Arg25 in
the coiled-coil trigger site resulted in a loss of helix content at
5 °C and an 18 °C decrease in Tm relative to
the wild-type protein (Table I). With the exception of a slight
difference in Tm, the results for the
GCN4p-E11A/R25A double mutant were similar to those for the GCN4p-R25A
single mutant.
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Fig. 1.
The surface salt bridge between
Glu22 and Arg25 stabilizes the GCN4 leucine
zipper dimer. A, sequences of the recombinant GCN4
proteins used in this study. The numbers refer to the amino
acid positions within GCN4p-1 (1). Heptad repeats are represented as
blocks of seven amino acid residues, and heptad positions are indicated
by lowercase letters. The 3,4-hydrophobic repeat with mostly
hydrophobic residues at heptad positions a and d
is indicated in bold. Residues that form intrahelical salt
bridges in crystal structures of GCN4 (1, 9, 10) are indicated by
arrows. The trigger site that drives coiled-coil formation
(2) is shaded in gray. B and
C, CD spectra recorded at 5 °C (B) and thermal
unfolding profiles (C) of GCN4p-wt (open
circles), GCN4p-E11A (closed circles), GCN4p-R25A
(open triangles), and GCN4p-E11A/R25A (closed
triangles). Thermal stability of the peptides was monitored by the
change of the CD signal at 222 nm. Polypeptide chain concentrations
were 75 µM in 10 mM sodium phosphate buffer
(pH 7.0) containing 150 mM sodium chloride.
Mean molar residue ellipticities, melting temperatures, and
molecular masses of the wild-type and mutant GCN4 leucine zipper
fragments and peptides
These findings demonstrate that the GCN4 leucine zipper coiled coil is
sensitive to the mutation in the trigger site. Both GCN4-pR25A and
GCN4p-E11A/R25A show sigmoidal thermal unfolding profiles
characteristic of a cooperative two-state coiled-coil to random-coil
transition. To further investigate the structural consequences of the
R25A mutation in the coiled-coil trigger site, 15N-labeled
GCN4p-wt and GCN4-pR25A proteins were examined by NMR. Chemical shift
indices for H resonances and 3JHNH
coupling constants measured from a three-dimensional HNHA experiment
are summarized for the wild-type and R25A mutant proteins in Fig.
2. Residues Lys3 through
Lys28 of the GCN4p-R25A mutant show negative H
chemical
shift indices (Fig. 2A) and 3JHNH
coupling constants below 6 Hz (Fig. 2B), consistent with
-helical structure (21, 23). The four C-terminal residues give H
and 3JHNH
values suggestive of
conformational averaging. The same conformational averaging is evident
for the four C-terminal residues in the wild-type protein (Fig. 2,
C and D). The differences in H
chemical shifts
and 3JHNH
coupling constants between
GCN4p-wt and GCN4p-R25A are summarized in Fig. 2, C and
D. In GCN4p-R25A, residues Val23 through
Lys27 flanking the R25A mutation, show small down-field
H
chemical shifts and increased 3JHNH
coupling constants relative to the wild-type protein. The changes in
these parameters suggest that the dynamic fraying of the C terminus in
the wild-type might extend toward the site of the R25A substitution in
the mutant. Nevertheless, the differences between the wild-type and
mutant are small, and the 3JHNH
and
H
chemical shifts for residues Val23 through
Lys27 in GCN4p-R25A remain within the limits for
-helical structure (23). On dissolving the GCN4p-R25A mutant in
D2O at pD 6.1, only two amide protons (Asn16,
and Glu32) are protected from hydrogen-deuterium
exchange after 12 min at 5 °C, and all amide protons are exchanged
after 40 min. In contrast, the 35-residue construct LZ, which has an
identical sequence to residues Met2 through
Glu32 of GCN4p-wt, shows much stronger hydrogen exchange
protection (24). At pD 6.2, the amide protons of residues
Met2 through Glu32 persist for 12 h and
the seven slowest exchanging amide protons from residues
Asn21 through Lys27 persist for at least 2 days
(24).
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Taken together, the NMR results suggest that the R25A substitution
induces only small differences in the -helix backbone structure of
GCN4p-R25A compared with GCN4p-wt. Based on the present data we cannot
exclude changes in side chain packing as a result of the mutation. The
extremely fast hydrogen exchange observed for GCN4p-R25A suggests that
whereas the
-helix backbone structure is conserved in the R25A
mutant, its stability is much lower than in the wild-type protein. This
conclusion is also supported by proteolytic susceptibility experiments
of the wild-type and mutant peptides. Whereas the GCN4p-wt was
resistant to trypsin cleavage at molar ratio of 10,000 to 1, GCN4p-R25A was completely digested after 2 h under the same
conditions (data not shown). These findings indicate that the entire
GCN4 leucine zipper dimer is destabilized by the R25A mutation in a
cooperative manner.
To assess whether monomer -helix stability within the GCN4 trigger
sequence is favored by the observed intrahelical salt bridge between
Glu22 and Arg25 in the leucine zipper dimer (1,
9, 10), we produced the two synthetic 16-residue peptides shown in Fig.
3. The GCN4p16-31 peptide corresponds to
the wild-type trigger sequence (2), whereas GCN4p16-31(R25A)
corresponds to the R25A mutation in which the putative salt bridge
between Glu22 and Arg25 is missing. As
previously reported (2), GCN4p16-31 is monomeric with significant
helicity (Fig. 3B; Table I). In contrast, the peptide with
the R25A substitution showed a 40% loss of helical structure according
to [
]222 (Fig. 3B; Table I). The
shift of the two minima at 205 and 222 nm in the CD spectrum also
indicate a substantial shift in the equilibrium from helix to random
coil.
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Helix formation of the GCN4p16-31 and GCN4p16-31(R25A) peptides was
further monitored as a function of salt concentration at physiological
pH. As shown in Fig. 3C, an increase in sodium chloride
concentration strongly disfavors -helical structure formation in the
wild-type peptide. This suggests that net electrostatic interactions
are favorable and helix-stabilizing. A decrease in
[
]222 with increasing salt concentration is also
observed for GCN4p16-31(R25A). The difference in helicity of the two
CD signals, however, indicates that a major component of the
interaction between Glu22 and Arg25 cannot be
screened by external salts. These findings suggest that the interaction
observed at high salt is not a simple electrostatic interaction and
that hydrogen bonding between the side chains of Glu22 and
Arg25 is a prominent determinant of the stability of
-helix monomer structure in the trigger site. This conclusion is
consistent with a recent study by Smith and Scholtz (25) who report
that in designed helical peptides the energetics of the interaction
between the fully charged ion pairs can be diminished by salt, but the interaction is not completely screened at high ionic strength.
We have previously reported that a helical trigger segment within the GCN4 leucine zipper monomer is indispensable for coiled-coil formation (2). Our findings demonstrated that this trigger sequence represents an autonomous helical folding unit. Our derived model proposed that productive coiled-coil formation of the GCN4 leucine zipper requires a contact of two trigger sequences at some stage in the folding pathway. Helical trigger sites are ideal for the initiation of coiled-coil formation because they ideally position the hydrophobic core residues and possible interhelical salt bridges for the recognition process. Moreover, such a mechanism would align the two chains in parallel register. Further folding of the coiled coil then proceeds in a zipper-like fashion from the C terminus and finally results in a stable leucine zipper dimer. This conclusion was in contrast to the GCN4 leucine zipper folding mechanism proposed by Sosnick and coworkers (26, 27) who reported a heterogeneous transition state that contains only little if any secondary structure. However, using diffusion collision theory, the folding data of Sosnick et al. (26) have been reinterpreted by Myers and Oas (3). These authors concluded that partial helix formation within the C-terminal part of the GCN4 leucine zipper monomer precedes dimerization, which is in line with our predicted folding model (2). Moreover, Zitzewitz et al. (4) subsequently carried out a detailed mutational and kinetic analysis of the GCN4 coiled coil. They found that the two C-terminal heptads are indeed the likely source of the nucleating helices and that helix-helix recognition between preformed elements of secondary structure plays an important role in the folding reaction.
Together, these considerations are consistent with the hierarchic view
of protein folding. Hierarchic protein folding is defined as a process
in which folding begins with structures that are local in sequence and
marginal in stability (28). These local structures interact to finally
fold into the native conformation. Accordingly, the GCN4 leucine zipper
monomer can be considered as an intrinsically disordered protein with a
marginally stable helical trigger site within its C terminus. Specific
recognition of two trigger sites induces folding of the unstructured N
terminus to finally yield the fully -helical coiled-coil dimer.
Consistent with the model of hierarchic protein folding, the synthetic
peptide GCN4p16-31 encompassing the coiled-coil trigger site is
~50% helical in the absence of tertiary and quaternary interactions
(Fig. 3). The intrahelical surface salt bridge between
Glu22 and Arg25 plays a key role in stabilizing
the helical structure of the coiled-coil trigger peptide. As a
consequence, introduction of the monomer helix destabilizing
mutation R25A, which does not affect the contact sites between helices,
resulted in destabilization of the GCN4 leucine zipper dimer without
causing significant changes in backbone structure (Fig. 2).
These findings are consistent with recent evidence. By introducing interactive combinations of intrahelical salt bridges into the GCN4 leucine zipper, Spek et al. (29) observed an increase in thermal stability of 22 °C of the mutant protein relative to the wild-type. As these mutations affect residues of the trigger sequence, a stabilizing effect on the monomer helix can be expected. Zhu et al. (30) reported that the effect on GCN4 dimer stability of an IIe to Asn mutation at the a position is greater at the N terminus of the peptide and decreases almost 2-fold as the substitution was moved toward the C-terminal heptads. This result supports the concept that monomer helix stability within the trigger site is more critical than the precise nature of the core residues. Finally, a hierarchical folding mechanism for coiled coils has recently also been suggested by Yu et al. (31).
A plausible explanation for the destabilizing effect of the R25A
mutation is an increase in the conformational entropy of the monomer
chains, which results in a reduction of the favorable entropic
contribution associated with GCN4 coiled-coil formation (32). In
contrast, only minor differences in enthalpies are anticipated for the
two proteins due to disruption of the salt bridge, because the contact
sites between helices are identical for the wild-type and mutant.
Therefore, a key function of the salt bridge between Glu22
and Arg25 is to limit the possible number of monomer chain
conformations and to provide an ideal scaffold for the interaction of
the critical core residues. This mechanism ideally allows the
two-stranded coiled coil to fold in parallel register. Consistent with
the concept of hierarchic protein folding, helical structure formation of the trigger site is determined largely by local sequence information.
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ACKNOWLEDGEMENTS |
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We are indebted to Dr. J. van Oostrum for support.
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FOOTNOTES |
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* This work was supported by grants from the Swiss National Science Foundation (to J. E. and A. T. A.).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.
§ Present address: School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK. To whom correspondence may be addressed: richard.kammerer@man.ac.uk.
Present address: Paul Scherrer Inst., Life Sciences, CH-5232
Villigen PSI, Switzerland. To whom correspondence may also be addressed: michel.steinmetz{at}psi.ch.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M010492200
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ABBREVIATIONS |
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The abbreviations used are:
NOESY, nuclear
Overhauser enhancement spectroscopy;
HMQC, heteronuclear multi quantum
coherence;
HSQC, heteronuclear single quantum coherence;
TOCSY, total
correlation spectroscopy;
wt, wild type;
HNHA, (amide
proton)-(nitrogen)-(-proton) correlation.
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