(Received for publication, September 12, 1995; and in revised form, November 30, 1995)
From the
The orientation of -helical chains in two-stranded
coiled-coils has been shown to be determined by the presence of
favorable interchain electrostatic interactions. In this study, we used de novo designed 35-residue peptides to show that when
interchain electrostatic interactions are not a factor in coiled-coil
formation, the relative positions of Ala residues in the middle heptad
can control the parallel or antiparallel orientation of
-helical
chains in coiled-coils. The peptides formed four-stranded coiled-coils
where the helices are either all-parallel or all-antiparallel with
respect to their nearest neighbor. The common structural element in
these four-stranded coiled-coils is an alternating pair of Ala and Leu
residues (Ala-Leu-Ala-Leu) in each of the two planes in the middle
heptad. These results indicate that both the relative positions of the
Ala residues in the hydrophobic core and the interchain electrostatic
interactions between charged residues in the e and g positions should
be considered in designing coiled-coils with the desired number of
strands in the multiple-stranded assembly. These design elements are
also important in orienting functional groups or domains attached to
the terminals ends of a coiled-coil carrier.
The simplicity and regularity of the coiled-coil has made this motif an excellent model for studying protein folding and protein-protein interactions. Unfortunately, the interactions that lead to the formation and stabilization of this ``simple'' motif are still not very well understood. The two basic objectives of a coiled-coil design are to attain the correct chain orientation, that is, parallel or antiparallel, as well as the desired number of helices in the coiled-coil assembly. The periodicity of hydrophobes in the a and d positions of the amino acid sequence of tropomyosin, as well as the charged residues in the e and g positions, have long been suspected to contribute to the packing and stability of two-stranded coiled-coils (Hodges et al., 1972; McLachlan and Stewart, 1975; Sodek et al., 1972; Stone et al., 1974). In the intervening years, a considerable effort has been spent in trying to understand how these two forces, hydrophobic packing and electrostatic interactions, determine the final folded structure.
On the one hand, there has
been an ongoing attempt to understand how the hydrophobic residues in
the coiled-coil interface control not only the stability of the
coiled-coil, but also the number of helices in the final coiled-coil
assembly. This research area has become tremendously important in light
of the present attempts to design multiple-stranded coiled-coils for a
variety of purposes, from synthetic catalysts to ligand carriers. The
general approach has been to vary the amino acid residues in the
hydrophobic core and to determine the structures of the resulting
analogs (Alber, 1992; Cohen and Parry, 1990; DeGrado et al.,
1989; Harbury et al., 1993; Hodges, 1992; Lovejoy et
al., 1993; Zhu et al., 1993). On the other hand, there is
a similar attempt to understand the role of interchain electrostatic
interactions in controlling the parallel or antiparallel orientation of
-helical chains in two-stranded coiled-coils. It has been shown
that the favored chain orientation is one that results in interchain
electrostatic attractions (Monera et al., 1993, 1994).
Presently, it is not known whether or not the type of hydrophobic
packing in the interface also controls the parallel or antiparallel
orientation of -helical chains in coiled-coils. In order to
address this question, we designed coiled-coils where interchain
electrostatic interactions are not a determinant of chain orientation
and, therefore, the formation of parallel or antiparallel coiled-coils
become mainly a function of the type of hydrophobic packing in the
interface.
To separate and purify the oxidized products, the peptide
solutions were neutralized with dilute acetic acid and then injected
into the Varian Series 5000 liquid chromatograph equipped with a
semi-preparative reversed-phase C column (Zorbax 300SB-C8,
9.4 mm
25 cm inner diameter, 5-µm particle size,
300-Å pore size). The samples were eluted at 2 ml/min with a
linear AB gradient of 1% solvent B/min for the first 15 min and 0.2%
solventB/min thereafter, where solvent A was 0.05% trifluoroacetic acid
in water and solvent B was 0.05% trifluoroacetic acid in acetonitrile.
Aliquots of the fractions were subjected to analytical HPLC and pure
fractions were pooled and lyophilized. In all cases, the identity of
the oxidized (disulfide-bridged) products were confirmed by amino acid
analysis, mass spectrometry, and HPLC characterization of the resulting
reduced peptides.
Circular dichroism spectroscopy was
performed at 20 °C on a Jasco J-500C spectropolarimeter (Jasco,
Easton, MD) calibrated with an aqueous solution of recrystallized
ammonium-d(+)-10-camphorsulfonic acid at 290.5 nm prior
to use (Monera et al., 1993). The standard deviations of
measurements at 220 nm was ± 300
degreescm
mol
.
Three important features were incorporated in the design of
starting peptides used in this study. First, the interchain
electrostatic interactions must be the same when the chains are
oriented either in a parallel or antiparallel manner. This requirement
was fulfilled by incorporating identical residues at the e and g
positions in the amino acid sequence of each peptide (Fig. 1).
Thus, two groups of peptides were designed and synthesized, one having
Glu at the e and g positions (the E peptides) and the other having Lys
(the K peptides). The cross-sectional diagrams of the middle heptads of
representative peptides 2E16 and 2K16 in -helical forms (Fig. 2, A and B, respectively) show that the
same potential interchain electrostatic interactions can occur in the e
and g positions whether the chains are viewed from the N or the C
termini. Although the type of electrostatic interaction is identical in
the parallel or antiparallel orientation, that is, with regard to
attractions or repulsions, structurally these interactions are not
necessarily identical. For example, when the heterodimer is parallel (Fig. 2E) the interactions involve g-e`/g`-e positions,
whereas when the heterodimer is antiparallel (Fig. 2F),
the interactions involve g-g`/e`-e positions. Molecular modeling of
parallel and antiparallel heterodimeric EK coiled-coils with Leu
residues in positions a and d were carried out as described previously
(Monera et al. (1993) and Lavigne et al.(1995)). In
both the parallel and antiparallel cases, owing to their flexibility,
the Lys and Glu side chains can bring their charged groups within
interionic distances (<5.0 Å) observed for salt bridges in
globular proteins (Rashin and Honig, 1984) by adopting staggered
dihedral angles (
i) frequently observed in globular proteins
(gauche minus (+60°) gauche plus (-60°), and trans
(180°), Morris et al.(1992)). Moreover, the amount of
buried hydrophobic surface area in the modeled and energy-minimized
parallel and antiparallel heterodimeric coiled-coils as measured with
the program ANAREA (Richmond, 1984; as implemented in VADAR version
1.2, Wishart et al.(1994)) is very similar. Accordingly, there
is no a priory reason to believe that g-e`/g`-e Glu-Lys salt
bridges in the parallel orientation are not energetically equivalent to
g-g`/e-e` salt bridges in the antiparallel orientation. In addition,
oppositely charged residues (Glu or Lys) are included at the f position
to increase solubility, but these residues are not capable of
intrachain electrostatic interactions with the oppositely charged
residues at the e or g positions.
Figure 1: Amino acid sequence of peptides used in this study. Each peptide contains five heptad repeats (35 residues) and the amino acid residues in each heptad repeat are designated by letters a-g, as indicated in the middle heptad. In the nomenclature used, the first digit(s) represent the position of the Cys residue (bold), the middle letter indicates the type of amino acids in both the e and g positions of each heptad repeat (boxed), and the last digits represent the position of the Ala residue (16 or 19) in the middle heptad (circled). The E peptides contain glutamic acid residues in the e and g positions and the K peptides contain lysine residues. The peptides are acetylated and amidated at the amino and C-terminal ends, respectively, in order to minimize charged interactions between the helical termini.
Figure 2:
Helical wheel diagrams showing the amino
acid residues in the middle heptad of the potential -helix from
reduced peptides (indicated by r) and potential coiled-coils
from oxidized peptides (indicated by x). A shows the
amino acid sequence of the middle heptad of 2E16r as viewed from the
C-terminal (CE, top) and N-terminal (NE, bottom)
ends, where E indicates that the peptide contains Glu at both
the e and g positions. The hydrophobic residue on the upper plane
(closer to the viewer) is boxed to emphasize its relative
spatial orientation relative to the hydrophobe in the lower plane. The
backbone arrows represent the direction of N
C chain
propagation. B shows the corresponding diagram for peptide
2K16r, where K indicates that the peptide contains Lys at both the e
and g positions. The rest of the conventions are identical to A. C represents the potential two-stranded
coiled-coil structure of the disulfide-bridged 2E16x homodimer as
viewed from the amino termini. The disulfide bridge is indicated as a bold line across the N termini of two helices (NE--NE). The two pairs of potential interchain
electrostatic repulsions per heptad are shown as dashed
arrows, for a total of 10 pairs for the two-stranded coiled-coil.
Hydrophobic packing in the upper and the lower planes are indicated as open and shaded double arrows, respectively. D, represents the 2K16x disulfide-bridged coiled-coil using
similar conventions used in C. The solid double arrows represent the two pairs of potential electrostatic attractions per
heptad, for a total of 10 pairs for the two-stranded coiled-coil. E-H are diagrams of the heterostranded coiled-coils
using similar conventions used in C and D. Note that in F the
coiled-coil is antiparallel and have opposite directions of chain
propagation.
Second, cysteine residues were
incorporated at either position 2 or 33 such that, after air oxidation
of appropriate peptide mixtures, the preferred products are
``trapped'' in the form of two-stranded oxidized
(disulfide-bridged) peptides. Thus, parallel coiled-coils are formed
when a disulfide bridge is formed between two cysteines at position 2
or two cysteines at position 33, while a disulfide bridge between
cysteines at position 2 and 33 would form an antiparallel coiled-coil.
We have previously shown that there are no structural restrictions in
the hydrophobic packing in forming antiparallel coiled-coils provided
that the interchain electrostatic interactions are appropriate (Monera, et al., 1993). Computer modeling studies based on an idealized
all-antiparallel four-helix coiled-coil indicate a distance of about
6-7 Å between -carbons in the a positions of chains
that are diagonally across or 3-4 Å between the two sulfur
atoms of cysteine residues. (
)Considering the flexibility of
the flared ends of the individual
-helical chains (Zhou et
al., 1992, 1993), these distances are within range for disulfide
bridge formation or at least require only minimal helical distortion to
form a disulfide bond. The insertion of an interchain disulfide bridge
at position 2a at the N-terminal of a two-stranded coiled-coil or
position 33d at the C-terminal do not introduce steric strain or
destabilize the structure (Zhou et al., 1993). In four-helix
coiled-coils the four helical chains are indistinguishable from each
other in terms of pitch and hydrophobic packing and the only
hydrophobes involved in the hydrophobic core are those of the three to
four hydrophobic repeat observed in two-stranded coiled-coils. In
contrast, a four-helix bundle refers to a more general definition where
the hydrophobic interactions between
-helices involve additional
hydrophobes other than those of the three to four hydrophobic repeat of
coiled-coils. The formation of the four-helix coiled-coil requires a
different set of hydrophobic interactions (Fig. 4) compared with
two-stranded coiled-coils (Fig. 2). In two-stranded parallel
coiled-coils positions ``a'' and ``d'' interact
with ``a`'' and ``d`,'' respectively, in the other
strand (Fig. 2). In four-stranded parallel coiled-coils position
``a'' of one strand (A16, Fig. 4C)
interacts with position ``a'' of two different strands (L16, Fig. 4C) and position ``d'' (L19, Fig. 4C) interacts with position
``d'' of two different strands (A19, Fig. 4C).
Figure 4: Helical rod schematic representation of the formation of disulfide-bridged products from air oxidation of peptides. The postulated four-stranded products are shown in dashed brackets. C and N represent the C- and N-terminal ends, respectively, and only one terminus of each chain is labeled for simplicity. The double arrows at the end of helices represent the potential disulfide bridge formation between cysteine residues. Potential hydrophobic interactions between Ala and Leu (indicated by double arrows) in two planes in the middle heptad are shown directly above their corresponding four-stranded coiled-coils.
Finally, Leu Ala substitutions were
made at either position 16 (a) or 19 (d) (Fig. 1) such that all
combinations of hydrophobic packings (Leu-Leu, Leu-Ala, and Ala-Ala)
were made possible in the middle heptad of the parallel two-stranded
coiled-coils, the antiparallel two-stranded coiled-coils, and the
different combinations of chain orientations in the four-stranded
coiled-coils. It should be noted that in all other heptads the
hydrophobic packing only involved Leu-Leu pairs. Therefore, because
there are no changes in the number and types of interchain
electrostatic interactions when two chains are aligned either parallel
or antiparallel to each other, the relative positions of the Ala
residues in the middle heptad are presumed to be the major (if not the
only) factor that determines the parallel or antiparallel orientation
of these coiled-coil assemblies.
All of the reduced E or K peptide analogs have been shown to exist as monomeric random coils under benign conditions, since coiled-coil formation would result in 10 pairs of interchain electrostatic repulsions at neutral pH (Fig. 2, C and D). In fact, air oxidation of individual E or K peptide analogs under benign conditions proceeded only very slowly to form two-stranded disulfide-bridged products, suggesting that the Cys residues are not pre-aligned by coiled-coil formation, and the disulfide bonds were formed mainly from random collisions of monomeric random coil species. The destabilizing effect of interchain electrostatic repulsions (Fig. 2, C and D) is so strong that even the stabilizing effect of the disulfide bridges (Zhou et al., 1993) of the 2E16x and 2K16x peptides is still not capable of maintaining a coiled-coil structure and the disulfide-bridged peptides only exist as two-stranded random coils (Zhou et al., 1994a).
Based on what is presently known about the factors that affect the formation and stabilization of two-stranded coiled-coils, we predicted that air oxidation of a mixture of 2E16r and 2K16r would spontaneously form the 2E16/2K16x disulfide-bridged heterostranded coiled-coil (oxidized heterodimer), considering that there are 10 pairs of favorable interchain electrostatic attractions (Fig. 2E). Surprisingly, such peptide mixture yielded very little heterodimer and, instead, gave the corresponding homodimeric oxidized products from each peptide (Fig. 3A). These oxidized homodimers were presumed to be unfavorable products because each has 10 pairs of unfavorable interchain electrostatic repulsions mentioned above. In contrast, air oxidation of the same peptide mixture under denaturing conditions (6 M guanidine HCl) yielded the 1:2:1 homodimer:heterodimer:homodimer (2E16x:2E16/2K16x:2K16x) ratio expected from random collision (data not shown). Finally, when a pure oxidized 2E16/2K16x peptide (formed under denaturing conditions and purified accordingly) was stirred in a benign redox buffer, only homodimeric oxidized products were found (Fig. 3B). These results were indeed puzzling, but suggest the possibility that under benign conditions some unknown structural elements were present that dictated the formation of the homostranded oxidized products from the reduced peptides. The formation of the products could be explained by the formation of a four-helix all-antiparallel coiled-coil, where the Cys residues react to form the disulfide bonds as indicated in Fig. 4A. Since the four-stranded coiled-coil is a noncovalent dimer, only the two-stranded disulfide-bridged peptides are observable as peaks in the reversed-phase HPLC chromatograms.
Figure 3:
Reversed-phase HPLC chromatograms of the
disulfide-bridged products of air oxidation of different peptides. The horizontal axes represent the retention times in minutes,
while the vertical axes represent the UV milliabsorbance
units. The specific reactions are schematically shown as insets, where only the relevant residues and positions are
indicated. For example, Cys is indicated at either position 2 (N
terminus) or 33 (C terminus), while Ala and Leu are at either position
16 or 19. The names of disulfide-bridged homo- and heterodimers are
indicated by an for oxidized
peptides.
In
order to verify further the presence of the postulated all-antiparallel
four-stranded assembly, we switched the cysteine residue of one of the
peptides from the N-terminal to the C-terminal position (33E16r) while
keeping all other design elements constant. When air-oxidized with
2K16r, the only product was the antiparallel heterodimer 33E16/2K16x (Fig. 3C). The same results were obtained when the
positions of cysteine were reversed, that is, when 2E16r was
air-oxidized with 33K16r, only the antiparallel heterodimer 2E16/33K16x
was produced (Fig. 3D). The formation of these
antiparallel heterodimers are possible only under two conditions: the
-helical chains in the assembly are all antiparallel with respect
to its nearest neighbor and the 4 cysteine residues have to be in the
same ends of the helices (Fig. 4B). Because of
proximity, disulfide bond formation would then be favored between
adjacent chains, which gives identical heterostranded antiparallel
products, rather than between opposite chains, which gives homostranded
parallel products. Taken together, all the results strongly indicate
the presence of a four-stranded, all antiparallel assembly when the
position of the Ala residues in the middle heptads are identical.
With the indication that an all-antiparallel, four-stranded
coiled-coil was the actual product formed from the mixture of 2E16r and
2K16r peptides, it became necessary to further investigate what was
controlling the formation of this all-antiparallel structure. Since by
design electrostatic interactions were essentially identical in either
parallel or antiparallel orientation, we then decided to vary the
position of the Ala residues in the middle heptads while keeping all
other design elements constant. This was done by switching the Ala
residue of the E peptide from position 16 to 19 (2E19r) and
air-oxidizing it with 2K16r. This time the product was a parallel
heterostranded peptide (2E19/2K16x, Fig. 3E), in direct
contrast to Fig. 3A. The exclusive formation of
2E19/2K16x can only be possible if the relative orientations of the
-helical chains in the four-stranded structure are reversed, that
is, from the all-antiparallel to the all parallel orientation, where
all cysteine residues are in the same end of the helices (Fig. 4C). This is also in direct contrast to the
arrangement of helices in Fig. 4A. Note that the
cross-section diagram of 2E19/2K16x (Fig. 2G) is very
similar to that of 2E16/2K16x (Fig. 2E), except for the
reversed designations of Ala and Leu at positions 16 and 19,
respectively, in peptide 2E19. Similarly, when mixtures of peptides
with reversed positions of the Ala residues (2E16 and 2K19) were
oxidized, only the parallel heterostranded 2E16/2K19x product (Fig. 2H) was observed (chromatogram not shown). As a
final test for this assumption, peptides 2E16r, 2E19r, 2K16r, and 2K19r
were mixed together and subjected to air oxidation. As expected, only
the parallel heterodimeric peptides (2E19/2K16x and 2E16/2K19x) were
the major products observed (Fig. 3F). These products
can only be exclusively formed when the relative orientations of the
-helical chains in the four-stranded structure are reversed, that
is, from the all-antiparallel to the all parallel orientation (Fig. 4C). The small amounts of homodimeric products
may have been formed from random collision and/or from excess peptides
due to small concentration differences of reduced peptides prior to air
oxidation. These were strong indications that the relative positions of
the Ala residues in the middle heptad control the parallel/antiparallel
or homodimer/heterodimer formation of products.
The CD spectra of
each disulfide-bridged 2E16x and 2K16x peptide under benign conditions
indicate a random coil structure (Fig. 5A), as reported
previously (Zhou et al., 1994a). However, when the two
peptides were mixed together either in reduced or oxidized forms their
negative molar ellipticities increased significantly (Table 1).
The disulfide-bridged 2E16/2K16x heterodimer also showed high molar
ellipticity. The approximately 1:1 ratio of their molar ellipticities
at 220 and 208 nm, coupled with the lack of significant increase in
molar ellipticity in the presence of 50% TFE (Fig. 5B),
indicate that these peptides assumed full coiled-coil structure under
benign conditions (Lau et al., 1984). Since each peptide in
the mixture is by itself not helical, the high ellipticity of the
mixtures indicate that the two peptides must be interacting in a
synergistic manner. This is consistent with our contention that the
actual product formed by the mixture of 2K16r and 2E16r under benign
conditions was a four-stranded coiled-coil. In the presence of TFE, the
disulfide-bridged 2K16x was induced to form full helical structure,
while 2E16x attained about 78% helical structure (Fig. 5B). The decrease in the ratio of the molar
ellipticities of these peptides in the presence of 50% TFE
(/
0.82) is more
characteristic of extended or noninteracting helices (Cooper and Woody,
1990; Lau et al., 1984).
Figure 5: CD spectra of the different peptide analogs under benign conditions (A) and in the presence of 50% TFE (B). The wavelength scans were done at 0.1-nm intervals and plotted at 2-nm intervals. The same symbols are used in A and B. See ``Materials and Methods'' for details.
SE ultracentrifugation and size-exclusion chromatography (SEC) experiments also support the postulated four-stranded assembly. For example, an equimolar mixture of reduced 2E16r and 2K16r appeared to be a mixture of dimers (two strands) and tetramers (four strands) from SE data and mostly tetramers from SEC results (Table 1). Similarly, an equimolar mixture of oxidized 2E16x and 2K16x were four-stranded from both SE and SEC data. Interestingly, the heterostranded 2E16/2K16x appeared to be two-stranded from both SE and SEC data. These results indicate that since 2E16x and 2K16x are random coils by themselves, the mixture of disulfide-bridged peptides are still capable of forming the four-stranded coiled-coil assembly. However, once the two-stranded coiled-coil is already formed, such as in 2E16/2K16x, it is too stable to open up to reform the four-stranded structure.
The most intriguing observation that led to this study was
the formation of only homodimers when a mixture of reduced peptides
2E16r and 2K16r were air-oxidized (Fig. 3A), which is
only possible if molecular aggregation, other than the two-stranded
form, exists. The postulated four-stranded, all-antiparallel
coiled-coil assembly that results from the mixture of 2E16r and 2K16r (Fig. 4A) is the only arrangement of -helices that
can explain the origin of the two-stranded disulfide-bridged products.
Coiled-coil forming peptides are thermodynamically favored to form
four-stranded coiled-coils because our molecular modeling studies (
)and x-ray crystallographic data (Harbury et al.,
1993) showed an increase of about 25-30% buried surface area in
going from the two-stranded to the four-stranded coiled-coil assembly.
In this all-antiparallel orientation, a disulfide bond can form between
the two cysteine residues across the four-helix structure (Fig. 4A). Thus, the disulfide-bridged homodimers were
observed upon separation by reversed-phase chromatography, even though
these homodimers cannot form two-stranded coiled-coil structures.
The results from the reoxidation of 2E16/2K16x heterodimer in redox
buffer (Fig. 3B) are also consistent with the oxidation
of reduced peptides, that is, only disulfide-bridged homodimers were
observed. The exclusive formation of disulfide-bridged homodimers
suggest that upon breakage of the disulfide bridges in redox buffer the
chains dissociate and reorient into the four-stranded, all-antiparallel
orientation described above. There are no other feasible packing
arrangements of the two-stranded forms that can lead to the formation
of mainly disulfide-bridged homodimers without dissociation of the
noncovalently bonded -helical chains. In addition, the
-helical chains in the 2E16/2K16x heterodimer does not appear to
open up under benign conditions, as evidenced by the SEC and SE results (Table 1). These results also demonstrate an example of two
proteins that by themselves exist in an unfolded form, but together
assemble into a specific folded structure.
The question arises, what
drives this orientation of -helical chains into an
all-antiparallel assembly? Theoretical calculations (Chou et
al., 1988; Hol, 1985; Hol et al., 1981) and previous data
(Monera, et al., 1993, 1994) suggest that, assuming everything
else being equal, the helix-dipole interactions favor the antiparallel
orientation. However, the results from this study strongly suggest that
since the positional effect of one Ala residue in the middle heptad is
capable of switching chain orientation from all-parallel to
all-antiparallel, or vice versa, the presumed contributions of
helix-dipole must be small.
If the -helical chains in the
four-stranded structure can switch between all-parallel to
all-antiparallel, what is the common structural element that determines
the chain orientation in all of these structures? Inspection of the
schematic diagram of the middle heptad of the 2E16/2K16x four-stranded
all antiparallel coiled-coil (Fig. 6A) shows that the
hydrophobic packing involves alternating Leu-Ala-Leu-Ala residues in
both upper and lower planes, as also shown in Fig. 6B.
Although predictably less stable than the Leu-Leu-Leu-Leu hydrophobic
packing, the Leu-Ala-Leu-Ala packing is stable enough to maintain the
four-stranded structure. If the
-helical chains were to orient in
an all-parallel manner, the hydrophobic packing would be
Ala-Ala-Ala-Ala in one plane and Leu-Leu-Leu-Leu on the other (Fig. 6C). Molecular modelling studies indicate that
when four Ala residues are aligned in one plane of a four-stranded
coiled-coil, stabilized by Leu residues on the other planes, the
resulting cavity was estimated to have a surface area of
177Å
and volume of 187Å
, large
enough to accommodate 5-10 water molecules.
The
alignment of four small and less hydrophobic Ala side chains (Zhou et al., 1994b) on the same plane of the four-helix coiled-coil
would create a large cavity that would be expected to destabilize the
four-stranded structure. Indeed, further inspection of the hydrophobic
packing in the middle heptads in Fig. 4reveals that the
four-stranded assembly invariably contains Leu-Ala-Leu-Ala in both
planes. The stability of the Leu-Ala-Leu-Ala packing was recently
demonstrated when analogs of the Rop protein where all the hydrophobes
in the helix interface were exclusively replaced with alternating
Leu-Ala packing were shown to exhibit native-like four-stranded
all-antiparallel structure (Munson et al., 1994).
Figure 6: Schematic representation of the interactions in the middle heptad of the postulated four-stranded coiled-coil for the mixture of 2E16 and 2K16 peptides (A). Viewing from the top, this diagram shows that the two N termini of 2E16 connected with a disulfide bridge (solid double arrow) and the C termini of 2K16. The disulfide bridge between the N termini of 2K16 is shown as a dashed double arrow (away from the reader). Similar conventions were used as in Fig. 2. B, schematic representation of the hydrophobic pairs in the two planes or levels in the middle heptad of the postulated all-antiparallel four-stranded coiled-coil and comparing those with an all-parallel orientation (C).
These
results clearly indicate that, at least in these peptide models, one
pair of alanine residues in the middle heptad can adequately control
the parallel or antiparallel orientation of -helical chains in the
four-stranded coiled-coil assembly. Multiple Leu
Ala
substitutions in the hydrophobic interface has minimal effect provided
that the hydrophobe on the other chain is a Leu or probably any of the
residues with large hydrophobic side chains. This positional effect of
alanine residues in the hydrophobic interface is important for at least
two reasons. One, this strategy can potentially be applied in
controlling the formation of two- and four-stranded coiled-coils.
Second, this design element may be critical in orientating two or more
functional groups or domains attached to a coiled-coil carrier.
Therefore, this positional effect of alanine residues in the
hydrophobic interface should be considered in conjunction with relevant
interchain electrostatic interactions between charged residues at the e
and g positions in the design of coiled-coils with predisposed
structures.