(Received for publication, June 5, 1995; and in revised form, July 31, 1995)
From the
The effects of interhelical electrostatic repulsions in
controlling the dimerization and stability of two-stranded
-helical coiled-coils have been studied using de novo designed synthetic coiled-coils. A native coiled-coil was
synthesized, which consisted of two identical 35-residue polypeptide
chains with a heptad repeat QgVaGbAcLdQeKf and a Cys residue at
position 2 to allow formation of an interchain 2-2` disulfide
bridge. This peptide, designed to contain no intrachain or interchain
electrostatic interactions, forms a stable coiled-coil structure at 20
°C in benign medium (50 mM KCl, 25 mM PO
, pH 7) with a [urea]
value
of 6.1 M. Five mutant coiled-coils were designed in which Gln
residues at the e and g positions of the heptad repeat were substituted
with Glu systematically from the N terminus toward the C terminus,
resulting in each polypeptide chain having 2, 4, 6, 8, or 10 Glu
residues. These substituted Glu residues are able to form interchain i to i`+5 electrostatic repulsions across the
dimer interface. As the number of interchain repulsions increases, a
steady loss of helical content is observed by circular dichroism
spectroscopy. The effects of the interchain Glu-Glu repulsions on the
coiled-coil structure are partly overcome by the presence of an
interchain disulfide bridge; the peptide with six Glu substitutions is
only 15% helical in the reduced form but 85% helical in the oxidized
form. The stabilities of the coiled-coils were determined by urea and
guanidine hydrochloride (GdnHCl) denaturation studies at 20 °C. The
stabilities of the coiled-coils determined by urea denaturation
indicate a decrease in stability, which correlates with an increasing
number of interchain repulsions ([urea]
values
ranging from 8.4 to 3.7 M in the presence of 3 M KCl). In contrast, all coiled-coils had similar stabilities when
determined by GdnHCl denaturation (approximately 2.9 M). KCl
could not effectively screen the effects of interchain repulsions on
coiled-coil stability as compared to GdnHCl.
The two-stranded -helical coiled-coil domain is an
important structural motif in a wide variety of proteins. These include
fibrous muscle proteins (McLachlan and Karn, 1982; Smillie, 1979) and
DNA-binding proteins, which are transcriptional regulators (Alber,
1992), as well as a host of other protein types (Adamson et
al., 1993; Cohen and Parry, 1990). The coiled-coil motif is
characterized by a heptad repeat denoted abcdefg, where a and d are
normally occupied by hydrophobic residues (Hodges et al.,
1972; Hodges, 1992), which fall on the same side of the helix,
resulting in a hydrophobic interface between the two helices which
provides the major driving force for formation and stability of the
coiled-coil (Hu et al., 1990; Zhou et al., 1992a,
1992b).
The positions e and g of the heptad repeat flank the hydrophobic face of these amphipathic helices and can participate in interhelical interactions, as well as shielding the hydrophobic core from water by folding across the dimer interface and making direct interactions with the hydrophobes in the core through the methylene groups of their side chains (Hodges et al., 1994; O'Shea et al., 1991). These positions generally contain charged residues, which may lead to interhelical electrostatic attractions or repulsions, thereby stabilizing or destabilizing the coiled-coil (Cohen and Parry, 1990; Talbot and Hodges, 1982). While many coiled-coils, including tropomyosin (McLachlan and Stewart, 1975; Stone et al., 1975) and transcriptional factors (Hu and Sauer, 1992; O'Shea et al., 1991), contain interhelical Lys-Glu and Arg-Glu salt bridges, which have been shown to stabilize them (Krylov et al., 1994; Zhou et al., 1994a), many coiled-coils have also been shown to be even more stable at pH 3, where protonation of the Glu residues prevents the formation of salt bridges (Lowey, 1965; Noelken and Holtzer, 1964; O'Shea et al., 1992; Zhou et al., 1994b). When more hydrophobic residues appear in these positions through random mutagenesis, more stable mutants were actually obtained (Hu and Sauer, 1992; Pu and Struhl, 1993; Schmidt-Dorr et al., 1991). Similar combinatorial mutagenesis studies (Hu et al., 1993) showed that GCN4 coiled-coils containing alanine and threonine at these positions were functional, also suggesting that the ion pairs were not critical.
Subsequently, interhelical ionic interactions between the e and g positions of coiled-coils have been shown to be more relevant in the specificity of coiled-coil formation. Recent studies (Baxevanis and Vinson, 1993; Graddis et al., 1993; O'Shea et al., 1993; Schuermann et al., 1991; Zhou et al., 1994b) have suggested that the presence of electrostatic repulsions in the homodimer will favor heterodimer formation if the number of interchain electrostatic repulsions can be reduced through heterodimer formation. This is the case for the Fos/Jun heterodimer, which forms preferentially by a factor of 1000 over the respective homodimers because both homodimers are destabilized relative to the heterodimer by interchain electrostatic repulsions as well as differences in interhelical hydrophobic interactions (O'Shea et al., 1992, 1989; Schuermann et al., 1991). In addition, electrostatic interactions have been shown to affect the chain orientation (parallel versus antiparallel) in model coiled-coils (Monera et al., 1994a, 1993). It is clear then that, in nature, fine tuning of the specificity of coiled-coil dimerization domains particularly in the DNA-binding proteins leads to a complex system of regulatory proteins each with a specific task (Jones, 1990; Ransone and Verma, 1990). In addition, other coiled-coils have been shown to form heterodimers, in particular tropomyosin (Lehrer et al., 1989; Lehrer and Stafford, 1991), and it is likely that interchain electrostatic interactions are important in dimerization specificity in these proteins as well.
In this paper we
take a detailed look at the role of electrostatic charge repulsion in
regulating the formation and stability of the -helical coiled-coil
dimerization motif, to obtain more insight into the importance of these
interactions in regulating the dimerization specificity of various
coiled-coil containing proteins in nature. Previous studies (Graddis et al., 1993; O'Shea et al., 1993; Zhou et
al., 1994b) have shown that many glutamic acid residues at the
positions e and g of model coiled-coil proteins prevented coiled-coil
formation, presumably due to the effects of interhelical charge
repulsion. Each Glu-Glu repulsion between a g and an e position has
been estimated to destabilize the coiled-coil by 0.45 kcal/mol (Kohn et al., 1995) and 0.78 kcal/mol (Krylov et al.,
1994). These repulsive destabilizing effects are over and above the
intrinsic destabilization of the Gln to Glu substitutions resulting
from differences in helical propensity and hydrophobic contributions to
the dimer interface and are the dominating effect (Kohn et
al., 1995). We would then predict that as the number of Glu
residues in these positions is varied, the degree of coiled-coil
formation should be altered significantly. This may be a function of
the overall net charge present in the dimer interface as well as the
effects of direct electrostatic repulsions. The present study uses a
stable coiled-coil containing zero net charge and no interhelical
electrostatic interactions at the dimer interface and systematically
increases the net charge as well as the number of potential specific
charge-charge repulsions by the substitution of glutamic acid residues
for glutamine to observe how the formation and stability of the
coiled-coil are affected. In particular, we are interested in
determining the degree of negative charge repulsion allowed before
coiled-coil formation is completely abolished. Finally, the ability of
salt and changes in pH to affect the degree of formation and the
stability of the model coiled-coil analogues is addressed.
where []
is the observed
ellipticity in millidegrees, MRW is the mean residue molecular weight
(molecular weight of the peptide divided by the number of amino acid
residues), c is the peptide concentration in mg/ml, and l is the optical path length of the cell in cm. Cell path lengths
were 0.05 cm for the data points in the denaturation studies and salt
and pH titrations and 0.02 cm for the CD spectra scans. CD spectra were
the average of four scans obtained by collecting data at 0.1-nm
intervals from 250 to 190 nm.
For monitoring the ellipticity at 220
nm, eight 1.0-s readings were averaged and repeated five times at each
data point and the resulting average was used (a total of 40 1.0-s
readings at each data point). Stock peptide solutions were prepared in
the appropriate buffer (0.1 M or 3 M KCl, 50 mM PO, pH 7 or pH 3). For urea and GdnHCl denaturation
studies, stock solutions of 10 M urea or 8 M GdnHCl
were prepared in the same buffer. The ratios of buffer and denaturant
solution added were varied to give the appropriate final denaturant
concentrations, and 10 µl of peptide stock solution was added to
each to make a total volume of 120 µl. Similarly, a stock 4 M KCl solution was used to make up the samples for the salt
titration of E10x. The pH titrations were done by making up separate
buffer solutions at the required pH values and adding 10 µl of
peptide stock solution to 110 µl of the buffers at each pH. The pH
of the final solutions for CD measurements were verified with a digital
pH meter. Peptide concentrations of the stock solutions were determined
by amino acid analysis as described previously (Kohn et al.,
1995), and an internal norleucine standard was added to each sample.
Since small errors in
the slope term (m) lead to large errors in the extrapolated
value of G
which gives the value of G
at the
denaturant concentration half-way between the
[denaturant]
values of the peptides.
Figure 1:
Amino acid sequences of the synthetic
peptides used in this study. Ac denotes N-acetyl, and amide denotes C
-amide. Native refers to the peptide
with all glutamine (Q) residues at positions e and g, shown in boldtype, of the heptad repeat, which is designated by the
letters abcdefg. In the peptide name, E stands for
substitutions of glutamic acid for glutamine, shown in boxes,
at positions e and g. The number following E is the
total number of glutamic acid substitutions in the analogue, in which
the substitutions proceed from the N terminus to the C terminus. All
peptides contain a single cysteine residue at position 2, allowing
formation of homodimers with an interchain disulfide bridge. The
resulting two-stranded peptides with a 2-2` disulfide bridge are
further designated with an ``x'' for oxidized and those
without the disulfide bridge are denoted with an ``r'' for
reduced.
The native sequence contains no interhelical or intrahelical electrostatic interactions. In a coiled-coil the e and g positions, which flank the hydrophobic interface made up of positions a and d, normally contain charged residues, which can participate in interhelical electrostatic interactions. In this sequence these positions contain neutral glutamine residues. Interhelical Lys-Glu interactions have been implicated as an important factor in stabilizing a parallel and in-register arrangement of the polypeptide chains (McLachlan and Stewart, 1975; Stone et al., 1975; Monera et al., 1993, 1994a; Zhou et al., 1994b) and were found to contribute about 0.4 kcal/mol per salt bridge to stability (Zhou et al., 1994a). However, the same study showed that replacement of the Lys and Glu residues with Gln led to a more stable coiled-coil despite the loss of the interhelical salt bridges. This is probably due to increased hydrophobic interactions at the dimerization interface between the side chains at positions e and g and the hydrophobic core a and d positions, which allow the hydrophobic core to be more sequestered from solvent.
In this study we introduced a cysteine residue at position 2 (position a in the heptad repeat) in order to form an interchain disulfide bridge, leading to a 70 residue two-stranded peptide. The disulfide bridge has the advantage of ensuring a parallel, in-register coiled-coil as well as removing peptide concentration as a determinant of the extent of coiled-coil formation and stability (Zhou et al., 1992c) since subunit association in solution is not a factor in the folding process when the two chains are covalently linked. When positioned at the N terminus of a 35 residue coiled-coil, a disulfide bridge has been shown to dramatically increase coiled-coil stability (Hodges et al., 1990; Zhou et al., 1992c, 1993).
The native sequence, designated N, was mutated by substitutions of glutamic acid for glutamine in a systematic fashion from the N terminus toward the C terminus as shown in Fig. 1. The resulting mutant peptides were designated E2, E4, E6, E8, and E10 according to the number of Glu residues at positions e and g in the 35 residue polypeptide chain (Fig. 1). The peptides with reduced cysteine at position 2 were designated with an ``r'' while those with an interchain disulfide bridge between cysteines at positions 2 and 2` were designated with an ``x.''
Shown in Fig. 2is a helical wheel diagram of the potential coiled-coil formed by E10. The diagram depicts with arrows the Van der Waals interactions between residues at the a and d positions on opposing chains as they pack together in the hydrophobic core and the potential electrostatic repulsions between glutamic acid residues at position e of one helix and at position g of the opposing helix. Although charged groups do not interact significantly in an aqueous environment, these residues are in a partially hydrophobic microenvironment since they are partially surrounded by hydrophobic residues. Therefore, despite being solvent-exposed, charged interactions between these residues can contribute to coiled-coil stability (Cantor and Schimmel, 1980; Krylov et al., 1994; Talbot and Hodges, 1982; Zhou et al., 1994a). The central boxes in Fig. 2containing the a, d, e, and g positions on chain 1 and the a`, d`, e`, and g` positions on chain 2 signify the complete dimerization interface (Adamson et al., 1993; Hodges et al., 1994; O'Shea et al., 1991) where any potential interhelical contacts are made.
Figure 2: Cross-sectional helical wheel diagram of coiled-coil E10. The direction of propagation of the polypeptide chain is into the page from N to C terminus with the chains parallel and in-register. This diagram depicts interchain a-a` and d-d` Van der Waals interactions between the hydrophobic side chains, which pack in a knobs into holes fashion (the prime indicates the corresponding position on the opposing helix). Also indicated are g-e` (i to i`+5) interchain repulsions between glutamic acid residues at position g of the heptad repeat on one helix and position e` of the following heptad on the other helix. The residues that comprise the entire dimerization interface at positions a, d, e, g, a`, d`, e`, and g` are within the centralboxes. Also given are the net charges on the nonpolar and polar faces of each helix (-10 and +5, respectively), as well as the net charge on each helix (-5) and on the dimer interface of the coiled-coil(-20).
The net charge at the dimer interface in this series of peptides ranges from zero in the native coiled-coil to -20 in the E10 coiled-coil as shown in Fig. 1.
The CD spectra of the peptides
are shown in Fig. 3. In panel A are the spectra for the
disulfide-bridged analogs Nx, E4x, E6x, E8x, and E10x in benign 25
mM PO, 50 mM KCl buffer at pH 7. The
double minima at 208 and 220 nm are characteristic of helical
structure. The CD band at 220 nm is due to the n to
*
transition and is responsive to the amount of helical content. The
predicted molar ellipticity for a 100%
-helical 35 residue
polypeptide was calculated as [
]
= -33,600 degrees
cm
dmol
(Hodges et al.,
1988) based on the theoretical equation of (Chen et al.,
1974). Therefore, Nx, with a [
]
of
-31,900, is highly
-helical (Table 1). The addition of
the helix-enhancing solvent TFE (Sönnichsen et
al., 1992) did not increase the helical content of Nx, as
indicated by the molar ellipticity at 220 nm (Table 1).
Figure 3: Circular dichroism (CD) spectra of the oxidized and reduced peptides. Spectra were recorded at 20 °C in a 25 mM phosphate, 50 mM KCl, pH 7 buffer. A, spectra for the oxidized disulfide-bridged peptides. B, comparison of reduced and oxidized peptides. Peptide concentrations are as follows. PanelA: Nx, 201 µM; E4x, 122 µM; E6x, 208 µM; E8x, 145 µM; E10x, 165 µM. PanelB: Nr, 203 µM; E4r, 243 µM; E6r, 306 µM; E4x and E6x same as panelA. Concentrations are given as the concentration of single stranded monomer for the reduced and two-stranded monomer for the oxidized peptides.
As shown in both Fig. 3A and Table 1, an increase in the number of glutamic acid residues per chain from 0 to 10 corresponding to an increase in the net charge at the dimer interface from 0 to -20 and an increase in the number of interhelical i to i`+5 Glu-Glu repulsions from 0 to 10, results in a gradual decrease in the amount of helical content from approximately 100% to 4%, which is indicative of a random coil structure. As mentioned above, the native coiled-coil Nx contains a high degree of helical content in benign conditions of low salt. E4x contains nearly the same helical content as Nx, so a net charge of -8 in the dimer interface has not significantly affected the coiled-coil structure. As the number of glutamic acid residues per chain is further increased to 6, 8, and 10, a progressive loss in helical content is observed until with 10 Glu residues/chain the amount of helical content is only about 4%. Therefore, the helical content has been systematically reduced through a gradual increase in the net negative charge at the dimer interface. This result is likely due to a shift in the folding equilibrium between folded and unfolded protein rather than a decrease in helicity of the folded state.
The increase in net
negative charge prevents dimerization primarily through interchain
electrostatic repulsions. However, this increase in negative charge has
a much smaller effect on the formation of monomeric -helices in
50% TFE. TFE has been shown to disrupt tertiary and quaternary
structure (Lau et al., 1984a). For example, the molar
ellipticity of E10x at 220 nm is increased from -1,500° in
benign buffer to -21,800° in 50% TFE (Table 1).
The
unfolding process for coiled-coils is not clearly understood, but the
most commonly accepted model is the two-state transition model, as
judged by the appearance of a monophasic denaturation curve. The
two-state model requires that a fully folded coiled-coil dimer unfolds
in one cooperative step to denatured monomers. Chain dissociation and
loss of helix are presumed to be simultaneous events (Engel et
al., 1991; Monera et al., 1994b; Skolnick and Holtzer,
1985; Thompson et al., 1993). This is consistent with the
observation that isolated -helices are generally unstable in
solution (Cohen and Parry, 1990; Dyson et al., 1988; Thompson et al., 1993).
Not all coiled-coils have been found to follow a two-state transition. In some cases biphasic denaturation has been observed (Greenfield and Hitchcock-DeGregori, 1993; Lehrer et al., 1989; Lehrer and Stafford, 1991), indicating that folding intermediates can occur as previously suggested by the ``continuum of states'' theory (Skolnick and Holtzer, 1986) in which a broad spectrum of partially folded intermediates may occur in the unfolding process. Therefore, while the simple two-state theory appears to adequately explain the unfolding process of most coiled-coils, it is not necessarily a universally correct description for the process.
In Fig. 3B and Table 1, the effect of reducing the interhelical disulfide bridge is illustrated. While the reduced native coiled-coil, Nr, is fully helical, the helical contents of E4r, E6r, and E8r are substantially decreased compared to their oxidized counterparts. A disulfide bridge between positions 2 and 2` has been shown to significantly stabilize model 35-residue coiled-coils (Hodges et al., 1990; Zhou et al., 1992, 1993). The increased stability of the coiled-coil resulting from the disulfide bridge is able to overcome the destabilizing effects of the interhelical charge-charge repulsions to a significant extent.
Figure 4:
pH dependence of the ellipticity of E10x
and E10r. All measurements were performed in 50 mM PO, 100 mM KCl buffer at 20 °C. A, CD spectra of E10x at pH 7, 6.25, and 5.4. B, mean
residue molar ellipticity at 220 nm versus pH for E10x and
E10r. Peptide concentrations were 43 and 156 µM for E10x
and E10r, respectively.
It is important to realize that the midpoint of the folding
transition curve is not the pK of the glutamate
side-chain carboxyl groups. In fact, the pK
values
of the glutamate side chains, which appear to influence the folding,
will not be constant but should be different in the folded and unfolded
states. If this were not the case, they would have little or no effect
on the pH dependence of stability (Yang and Honig, 1993). Therefore, if
an unfolding transition takes place over a certain pH range, the
pK
value(s) of the group(s) responsible for
causing the transition does not correspond to the pH at the transition
midpoint, but instead the pK
of these groups is
shifted from one value that corresponds to the titration start point
(pK
in the folded protein) to another value that
corresponds to the titration end point (pK
in the
unfolded protein) (Yang and Honig, 1993). The correlation between the
transition end points and the pK
values will not
be exact, especially when more than one ionizable residue is
responsible for the pH dependence.
The pH-induced conversion from random coil monomer to coiled-coil dimer was also shown by the size-exclusion chromatograms of E8r (at a flow rate of 0.2 ml/min to increase resolution) at different pH values ranging from 7 to 5 (Fig. 5). At pH 7 peptide E8r elutes at about 56 min, corresponding to the monomer. As the pH is reduced, a peak at about 51 min due to the coiled-coil dimer begins to appear by pH 6. At pH 5.87, there are two approximately equal peaks in the chromatogram, indicating that this is the midpoint of the conversion. As the pH is further reduced, the dimerization becomes complete by pH 5.5. This pH-induced folding is very similar to that observed by circular dichroism in Fig. 4. In both cases, the folding transition is rapid, taking place within one pH unit, and the midpoints are similar. For E10r the apparent pH transition midpoint was 5.6 using CD, while for E8r the apparent pH transition midpoint was 5.87 using SEC. Thus, there is a correlation between the transition in helicity as measured by CD and subunit association as measured by SEC, which supports a two-state transition from helical dimer to random coil monomers.
Figure 5:
Effects of pH on the conformation of E8r
as monitored by size exclusion chromatography. All runs were carried
out in 50 mM PO, 100 mM KCl buffer with a
flow rate of 0.2 ml/min on a Pharmacia Superdex 75 column. The peptide
was dissolved in running buffer containing 10 mM DTT to keep
it reduced. An internal standard 10 residue unstructured peptide with
the sequence Ac-RGAGGLGLGK-NH
was included in each run to
confirm run to run reproducibility. M and D represent
the monomeric and dimeric forms of peptide E8r,
respectively.
The
preference for the order of association of a coiled-coil has been shown
to be dependent mainly on the residues in the a and d positions of the
heptad repeat. Val at position a and Leu at position d, as in our
sequences, have been shown to favor dimer and trimer formation over
tetramer formation (Harbury et al., 1993; Lovejoy et
al., 1993; Lupas et al., 1991). Recent studies with the
GCN4 leucine zipper showed that a mutant with Val at all a positions
and Leu at all d positions formed a mixture of dimer and trimer
(Harbury, et al., 1993). Another study where the asparagine at
position 16 (position a of the heptad repeat) of the 33-residue leucine
zipper of GCN4 was substituted with valine yielded a triple-stranded
-helical coiled-coil with a temperature denaturation midpoint 40
°C higher than the dimeric native sequence (Potekhin et
al., 1994). Their conclusion was that having Val at a and Leu at
position d favors trimer structure but that the presence of a polar
group such as asparagine in the a and d positions directed two-stranded
coiled-coil formation instead.
Size-exclusion chromatography of the series of coiled-coil analogues in both the reduced and oxidized forms were carried out (data not shown). In the case of the oxidized peptides, all six eluted in a narrow range of retention times predicted from the standard curve to correspond to the two-stranded 70-residue monomer. In the case of the reduced analogues, Nr, E2r, and E4r were predicted by SEC to be present as dimers, while E6r, E8r, and E10r were predicted by SEC to be present as monomers, corresponding with the results of the CD spectral analysis.
Sedimentation equilibrium
experiments in the analytical ultracentrifuge were carried out as
described previously (Kay et al., 1991; Zhu et al.,
1993) to confirm the assignment of monomeric and dimeric species. The
E8r peptide gave an apparent M of 3600 (Fig. 6), which closely matches the calculated M
of 3696 for the monomer. This confirms our assignment of E6r, E8r
and E10r, which coeluted in SEC, as monomers. Similarly, an apparent M
of 7790 was determined for Nr (Fig. 6),
which closely agrees with the expected dimer M
of
7376.
Figure 6:
lnYversusr plot from sedimentation equilibrium
experiments on Nr and E8r. Samples were run in 50 mM PO
, pH 7 buffer with 10 mM DTT. The buffer
contained 100 mM KCl for E8r and 1 M KCl for Nr. Both
samples were brought into equilibrium at a rotor speed of 44,000 rpm in
a Beckman model E analytical ultracentrifuge. Concentrations were 1.08
mg/ml (290 µM) and 0.80 mg/ml (216 µM) for
E8r and Nr, respectively.
Further evidence for the association state was obtained by use of a low angle laser light scattering detector (Arakawa et al., 1992) to estimate molecular weights of the peptides eluted from a Superdex 75 size-exclusion column. The results gave a molecular weight for E4r twice that for E6r, which indicates that E6r is present as the monomer and E4r is present as the dimer, in agreement with the assignment of association state by retention time.
We conclude from size-exclusion chromatography, ultracentrifugation, and laser light scattering that our sequence preferentially forms a two-stranded coiled-coil. Thus, either the cysteine at position a in the reduced coiled-coils or sequence differences at other positions could be destabilizing trimers, analogous to the effect of the buried Asn in GCN4. Recent studies by Krylov et al.(1994) and Alberti et al.(1993) have shown that residues in the positions e and g of the heptad repeat can greatly affect the order of association.
Figure 7:
Denaturation profiles of some of the
disulfide-bridged coiled-coils at 20 °C in 50 mM PO, 100 mM KCl buffer using: urea at pH 7 (A), GdnHCl at pH 7 (B), and GdnHCl at pH 3 (C). The fraction of folded peptide was calculated from the
observed mean residue ellipticity at 220 nm, as described under
``Materials and Methods.'' For those analogues that are not
fully helical, the fraction folded was calculated based on the
ellipticity of Nx in benign conditions. The peptide concentrations in
the final solutions for CD measurements ranged from 70 to 100
µM.
The
[urea] for Nx is 6.1 M, indicating
significant stability in the absence of interhelical electrostatic
interactions and making this coiled-coil a good control with which to
compare the mutant analogs (Fig. 7A, Table 2).
E4x has a [urea]
of 5.1 M and a
G
value of -0.8 kcal/mol. With the
addition of another 2 glutamic acid residues/chain in E6x the decrease
in stability as compared with that of Nx is more pronounced, with a
[urea]
of 2.8 M and a
G
of -2.3 kcal/mol. E8x is only
about 50% folded under benign conditions, so its
[urea]
by definition is 0 M and it has
a
G
of -4.4 kcal/mol. Finally, the
stability of E10x cannot be determined under these conditions (pH 7, 20
°C) since it is too unstable to show appreciable helical content.
As was observed for the amount of helical content, the increased charge
repulsion at the coiled-coil interface has substantially decreased the
[urea]
in a progressive fashion.
The GdnHCl
denaturation profiles at pH 7 (Fig. 7B) show a much
different result from those obtained with urea. The
[GdnHCl] values for Nx, E4x, E6x, and E8x are
all very similar, in the range from 3.1 M to 2.8 M (Table 2). The results with GdnHCl do not reflect the large
systematic decrease in the stability of the coiled-coils that was
observed with urea denaturation as the net interface charge and degree
of interhelical charge repulsion is increased. These observations
support previous results from this laboratory (Monera et al.,
1994a, 1994b, 1993), which showed that GdnHCl appears to mask
electrostatic repulsions and attractions in two-stranded coiled-coils,
giving the same measure of stability whether the residues at positions
e and g were arranged to form interhelical attractions or repulsions as
long as the hydrophobic packing at the dimer interface was the same.
Similarly, Hagihara et al. (1994) have also recently
illustrated this same phenomenon, where urea denaturation at pH 7
showed progressively lower stability as the degree of acetylation of
horse heart cytochrome c was increased while GdnHCl
denaturation gave the same stability regardless of the extent of
acetylation. This indicates an ability of GdnHCl to mask the negatively
charged residues and their interactions with other charged residues
that contribute to stability. The ionic nature of GdnHCl is the
probable reason for its ability to disrupt the effects of charged
residues on protein stability. The positively charged guanidinium ion,
which is responsible for binding to the protein surface and subsequent
denaturation (Makhatadze and Privalov, 1992; Greene and Pace, 1974;
Pace, 1986; Tanford, 1970), may initially bind specifically at the
negatively charged glutamate carboxyl groups when present at low
concentrations, thereby neutralizing the charge repulsions and removing
their effects on stability.
At low pH, the stabilities of many
coiled-coils have been observed to be higher than at pH 7, as shown for
example in the muscle protein tropomyosin (Lowey, 1965; Noelken and
Holtzer, 1964), the Fos-Jun heterodimeric leucine zipper (O'Shea et al., 1992), and synthetic model coiled-coils (Hodges et
al., 1994; Zhou et al., 1992c, 1993). Zhou et al. (1994a) demonstrated that a protonated Glu residue (at pH 3) at
the e position of the heptad repeat in a coiled-coil makes a 0.65
kcal/mol greater contribution to coiled-coil stability than an ionized
Glu residue (at pH 7) and a 0.45 kcal/mol greater contribution to
stability than a Gln residue in the absence of interhelical or
intrahelical charge-charge interactions. Recently, Lumb and Kim(1995)
have found that the pK of a Glu residue at
position 20 of the GCN4 leucine zipper (position e of the heptad
repeat) is slightly higher in the folded state than in the unfolded
state, also indicating that the protonated form is energetically
favorable. Our study supports these results as shown in Fig. 7C, where the GdnHCl denaturation profiles at pH 3
show that as the number of protonated Glu residues is increased the
stability of the coiled-coil is increased, with Nx being least stable
and E10x the most stable at pH 3. Nx has the same stability as at pH 7,
as one would expect, since there are no residues that change their
ionization state over the pH range 3 to 7. E10x is so stable at pH 3
that it is only about 50% unfolded in 7.4 M GdnHCl (the
maximum experimentally possible concentration). This peptide is fully
folded in 9 M urea (data not shown), indicating a greater
efficiency of GdnHCl as a denaturant versus urea as described
previously (Greene and Pace, 1974). Protonated Glu residues have been
shown to have higher helical propensity than Gln (Chakrabartty et
al., 1994; Scholtz et al., 1993), and it has been shown
that helical propensities of side chains can affect coiled-coil
stability (Hodges et al., 1981; O'Neil and DeGrado,
1990). In addition, the higher hydrophobicity of protonated Glu
compared to Gln (Guo et al., 1986; Sereda et al.,
1994) can increase coiled-coil stability when located at positions e
and g. The side chains at these positions can extend across the
hydrophobic interface, thereby reducing solvent accessibility to the
hydrophobic core. More hydrophobic residues in these positions have
therefore been shown to lead to higher coiled-coil stability
(Schmidt-Dorr et al., 1991; Zhou et al., 1994a;
Hodges et al., 1994).
Figure 8:
Effects of KCl and GdnHCl on the helicity
of peptides Nx and E10x. A, comparison of the effects of
GdnHCl on the structures of peptides Nx and E10x in the presence of 50
mM PO, 100 mM KCl, pH 7 buffer at 20
°C. The fraction folded peptide was calculated as under
``Materials and Methods.'' Peptide concentrations were 89
µM for Nx and 82 µM for E10x. B,
effects of KCl and GdnHCl concentration on the mean residue molar
ellipticity of E10x. All measurements were performed in a 50 mM PO
, pH 7 buffer at 20 °C. Peptide concentration
was 73 µM in the GdnHCl titration and 67 µM in the KCl titration.
We have compared the abilities of
GdnHCl and KCl to induce helical structure in the E10x peptide as shown
in Fig. 8B. In the case of GdnHCl, the maximum
inducible helix (-29,000
degreescm
dmol
) is achieved
at about 0.75 M and is about 90% of the helical content of Nx.
KCl is able to induce the same degree of helical structure but requires
a concentration of over 2 M to do so. The potassium ion has
the same plus one charge as the guanidinium ion but does not have the
same apparent binding affinity for the glutamate groups. We propose
that the guanidinium ion can bind to the glutamate groups via an
electrostatic interaction and through hydrogen bonding while potassium
ion can only bind to the glutamate carboxyl group through an
electrostatic attraction. Therefore, the added effect of hydrogen
bonding may increase the overall affinity of the guanidinium ion for
the glutamate carboxyl groups and thereby increase its ability to mask
the charge repulsions. The ability of salts to have general
Debye-Hückel charge screening effects should depend on the ionic
strength and therefore be the same for equal concentrations of GdnHCl
and KCl. Thus, general charge screening appears not to be the major
contribution to the induction of helix in E10x by GdnHCl.
It has long been accepted that salts can affect hydrophobic interactions in proteins. KCl promotes protein stability mainly through its effect on the structure of water, which increases the stability of the folded state by decreasing the solubility of hydrophobic molecules and increasing the apparent hydrophobic effect (Creighton, 1993). This type of salt is predicted to be excluded from the protein surface, meaning that its concentration is lower around the protein molecules than in the bulk solvent, and the protein is described as preferentially hydrated. In contrast, a denaturing salt such as GdnHCl tends to increase protein solubility (including the hydrophobic core) via direct interactions of the guanidinium ion with the surface of the protein, as proposed in the denaturant binding model (Makhatadze and Privalov, 1992). The binding of the denaturant to the protein surface allows the exposure of the normally buried hydrophobic core and a much greater surface area of the protein exposed to solvent. Thus, the different apparent abilities of KCl and GdnHCl to promote the E10x coiled-coil structure may be due to different mechanisms of action by the two salts; GdnHCl potentially operates mainly by masking charge repulsions through direct ion binding, whereas KCl may act more through promoting a stronger hydrophobic effect that can override the effects of the charge repulsions on the coiled-coil folding (i.e. it is able to force the coiled-coil to form even in the presence of the charge-charge repulsions).
Possible evidence for this line of
reasoning is given by the results of urea denaturation of the
disulfide-bridged coiled-coils at pH 7 in the presence of 3 M KCl (Fig. 9A). The large KCl concentration has
shifted the stabilities of the entire series of analogues
significantly, to higher [urea] values. As a
result, the stabilities are now such that even the lowest stability
analogues E10x and E8x are almost fully helical in the absence of urea (Table 1), and their entire denaturation with urea can be
observed and compared with that of the other analogues. The shifting of
these denaturation curves by the presence of 3 M KCl clearly
illustrates the ability of KCl to increase the apparent hydrophobic
effect. This effect on stability should be the same for all the
analogues since they share the same hydrophobic core residues and is
seen clearly from the increase in the [urea]
of
Nx (which is not affected by electrostatic interactions) from 6.1 M to 8.4 M (Table 2) as the KCl concentration is
increased from 0.1 M to 3 M. This corresponds to an
increase in the
G
(free energy of unfolding)
of 1.8 kcal/mol. This extra stabilization is therefore able to
counteract the destabilizing effects of the interchain repulsions
enough to promote full coiled-coil formation. There may also be some
degree of charge screening by the KCl contributing to the increased
coiled-coil formation and stability of the analogues with repulsions,
but this effect is not large since a wide range of stabilities (range
in [urea]
from 3.7 to 8.4 M; see Table 2) is obtained in the high KCl concentration. If KCl were
very effective at screening the charge-charge repulsions, all the
analogues should have similar [urea]
values.
Similar results were obtained by Monera et al.(1993) where the
inability of KCl to screen electrostatic interactions was also
observed.
Figure 9:
Effects of KCl and pH on the apparent
stability of the disulfide-bridged coiled-coils in the presence of urea
and GdnHCl. A, urea denaturation profiles at 20 °C in 50
mM PO, 3 M KCl buffer at pH 7. The
fraction folded was calculated as described under ``Materials and
Methods.'' The peptide concentrations in the final solutions for
CD measurements ranged from 72 to 108 µM. B,
G
versus the number of Glu
residues per chain under different denaturation conditions. Results
shown are for GdnHCl denaturation in 50 mM PO
, 100
mM KCl buffer at pH 7 and 3, and for urea denaturation in 50
mM PO
, 3 M KCl buffer at pH 7. All
measurements were done at 20 °C, and
G
was calculated as outlined in Table 2.
In contrast to KCl, GdnHCl does not increase protein
stability through promoting stronger hydrophobic interactions. For
example, the urea denaturation of Nx in the presence of 1 M GdnHCl gave a [urea] of 4.8 M (data not shown) versus 6.1 M without the GdnHCl
present. This corresponds to the known effects of GdnHCl on decreasing
the temperature stability (T
) of proteins (Von
Hippel and Wong, 1965).
In contrast to the results of the urea
denaturation, the denaturation in GdnHCl at pH 7 shows that
G
is essentially unaffected (Fig. 9B), demonstrating the ability of GdnHCl to
screen the effects of the electrostatic repulsions on coiled-coil
stability. At pH 3 the stability increases (a positive
G
, indicating the coiled-coil is more
stable than the native coiled-coil) with the number of Glu residues per
helix as indicated by Fig. 7C. In the middle part of
the coiled-coil (going from 2 to 8 Glu residues/chain), there is a
linear increase in stability of about 1.8 kcal/mol per increase of 2
Glu residues/chain (four in the dimer). This corresponds to 0.45
kcal/mol increase in stability per Gln to protonated Glu substitution,
which is consistent with the study of Zhou et al. (1994a) in
which protonated Glu was substituted for Gln only at the e positions.
Therefore, the substituted Glu residues appear to have equal effects on
stability at both the e and g positions of the heptad repeat.
The addition
of 3 M KCl to the solution brings the [urea] value for E2x back below that of Nx (Fig. 9A),
indicating that the charge-dipole interaction is screened by high salt
concentration. This result indicates that KCl is able to screen
charge-dipole interactions even though it could not effectively screen
the charge-charge interactions between Glu side chains as shown in Fig. 9A, suggesting that the charge-charge interaction
is stronger. Similarly the positioning of negatively charged residues
at the C-terminal end of the coiled-coil is likely to have a
destabilizing effect due to an unfavorable interaction with the
negatively charged end of the helix macrodipole, and a high salt
concentration should screen this interaction. This may explain why
there is a large decrease in helicity between E8x and E10x under low
salt conditions (Fig. 3A), but there is not a
significant difference in the stability of E8X and E10x by urea
denaturation in the presence of 3 M KCl (Fig. 9A) where charge-dipole effects are screened.
The model coiled-coils from our previous study (Kohn et al., 1995) serve as key controls for this paper since they unequivocally show that an interchain Glu-Glu repulsion in the i to i`+5 orientation destabilizes a coiled-coil by 0.45 kcal/mol. We have subsequently shown in the present study that a series of coiled-coils containing a systematic increase in the number of such Glu-Glu repulsions result in a gradual loss of helical content and stability. The fact that the helical content and stability decrease gradually indicate that the repulsive effects introduced by Glu substitutions are additive. While there is not a critical point at which an all helical coiled-coil is transformed to a random coil by the substitution of two additional Glu residues, the loss of helical content is significant in going from E6x to E8x in the oxidized coiled-coils and from E4r to E6r in the reduced coiled-coils, indicating the importance of subtle changes in interchain electrostatic interactions in determining coiled-coil formation, specificity, and stability.
The question still remains as to the mechanism of destabilization of the coiled-coil by the interchain repulsions. As stated by Zhou et al. (1994b), interhelical electrostatic repulsions can disrupt the formation and stability of coiled-coils in two ways. First, specific i to i`+5 (g-e`) repulsions between two like-charged side chains could destabilize the coiled-coil. The repulsion between two like-charged residues on opposing helices of the coiled-coil may not destabilize the coiled-coil through the electrostatic repulsion forcing the chains apart, since to do so, it would have to overcome the much more significant hydrophobic effect, which is the major force driving coiled-coil formation. Normally the side chains that occur in the e and g positions lie across the dimer interface and interact with each other forming salt bridges when they are oppositely charged; however, when they are similarly charged, the repulsions between the side chains may force them apart and away from the dimer interface. This would leave the hydrophobic core a and d positions more exposed and therefore lead to a decrease in stability by allowing greater solvent access and therefore reducing solvent entropy in the folded state. Second, the buildup of a large net charge on the dimer interface could also lead to general electrostatic effects in which net charges on the faces of the two helices repel.
Determining the relative roles of these two effects is somewhat
difficult from the current understanding of interhelical electrostatic
effects on coiled-coil formation and stability. However, we have found
that a coiled-coil containing 5 interhelical Glu-Glu and 5 interhelical
Lys-Lys repulsions but zero net charge at the dimer interface forms a
coiled-coil in both the case of parallel and antiparallel chain
orientation (Monera et al., 1993). ()In contrast
peptides containing 10 interhelical Lys-Lys or 10 interhelical Glu-Glu
repulsions with high net charges at the prospective dimer interface
will not form coiled-coils (Graddis et al., 1993; Zhou et
al., 1994b; this study). In addition, a synthetic triple-stranded
coiled-coil with a net charge of -3 in the trimer interface forms
despite seven pairs of specific interhelical ionic repulsions between
residues at the e and g positions of the helices, which lie
antiparallel to each other (Lovejoy et al., 1993). Therefore,
the high net charge may be preventing the chains from approaching each
other through general electrostatic repulsions while specific charge
repulsions between these residues may destabilize the coiled-coil by
disrupting side chain packing around the dimer interface. However, the
relative roles of these two forms of electrostatic effects will
probably depend on the surrounding amino acids.
The effects of disulfide bridges on the stability of two-stranded coiled-coils have been described previously (Hodges et al., 1990; Zhou et al., 1993). In the present study, we have illustrated the ability of disulfide bridges to overcome the effects of interhelical charge-charge repulsion on coiled-coil formation ( Fig. 3and Fig. 4). While the effect of the disulfide bridge on stability is difficult to interpret because it involves the comparison of a bimolecular concentration-dependent unfolding process for the reduced coiled-coil and a unimolecular concentration-independent unfolding process for the oxidized coiled-coil (Regan et al., 1994), we have estimated the interchain disulfide bridge to offer about 3-4 kcal/mol additional stability to the coiled-coil (Hodges et al., 1990; Kohn et al., 1995). This effect would be expected to be capable of counteracting a large number of interchain electrostatic repulsions, which as stated above have been estimated to destabilize by 0.45 kcal/mol per Glu-Glu repulsion. In the case of the E8x peptide with a net charge of -16 and containing 8 interhelical Glu-Glu repulsions, the disulfide bridge is able to promote 50% coiled-coil structure. Therefore, in this case the stabilizing factors including the disulfide bridge are being almost offset by the charge repulsion so that the peptide is equally populating the folded and unfolded states.
Although the disulfide
bridge does not generally apply to natural coiled-coils, there are some
cases where redox control of transcription factor activity in
eukaryotic cells has been suggested. Recently it has been found that
the basic helix-loop-helix transcription factor E2A binds DNA at
physiological temperature as a homodimer only in the presence of an
interhelical disulfide bond, while under reducing conditions E2A binds
DNA only as a heterodimer with MyoD or Id (Benezra, 1994). Nuclear
translocation of the transcription factor NF-B is activated by
oxidative stress (Meyer et al., 1993; Schreck et al.,
1991), while USF (a basic helix-loop-helix zipper protein) forms both
intra- and inter-molecular disulfide bonds, which appear to decrease
its affinity for DNA (Pognonec et al., 1992). Therefore, the
fact that an intermolecular disulfide bond is required for high
affinity DNA binding of certain transcription factors and that the
presence or absence of this bond can profoundly direct dimerization
specificity at physiological temperatures suggests that, as observed in
this study, the ability of the disulfide bridge to overcome
destabilizing effects on homodimerization such as intermolecular charge
repulsion is the key to its role in transcription factor activity.
The dramatic effects of pH and salt on protein folding observed in this study and previously in model coiled-coils (Zhou et al., 1994b) and other model helical proteins (Ramalingam et al., 1992) have suggested major implications for the potential of de novo design of environmentally sensitive proteins. One of the best examples of salt effects on protein folding in native proteins is the case of extreme halophilic bacteria, which are adapted to living in high salt concentration environments and whose cytoplasm is close to saturated in KCl. The proteins of these bacteria are often rich in acidic groups and have lower hydrophobicity than their counterparts in non-halophilic bacteria (Lanyi, 1974). These proteins, when isolated, require high salt concentrations to stabilize their folded structures. This stabilization may be due to the combined effects of the salt stabilizing the hydrophobic core as well as interactions of hydrated salt ions with the surface of the folded protein (Zaccai and Eisenberg, 1990). It has been suggested that clustering of negatively charged residues on the surface of halophilic proteins may cause structural instability, possibly due to charge repulsion, that is removed by the effects of salts (Ramalingam, et al., 1992). pH sensitivity of coiled-coil formation has recently been illustrated for the influenza virus hemagglutinin protein, which is required for fusion of the viral and cellular membranes. The protein undergoes a conformational shift to become active under the mildly acidic (pH 5) conditions of the mature endosome (Carr and Kim, 1994). The protein forms a three-stranded coiled-coil stem adjacent to a sequence of about 35 residues, which forms an extended loop at pH 7 but at pH 5 becomes helical and extends the triple-stranded coiled-coil, thereby inducing the activating changes in the structure. It has been found that this sequence probably forms an extended loop at pH 7 due to interchain electrostatic repulsion, which prevents coiled-coil propagation through this region but which is alleviated by protonation of acidic side chains at lower pH (Carr and Kim, 1993).
In conclusion, this study illustrates that a systematic increase in interhelical charge repulsion leads to a progressive loss of helical content and stability. These effects can be modulated by changes in pH and high salt concentrations.