Observation of glycine zipper and unanticipated occurrence of ambidextrous helices in the crystal structure of a chiral undecapeptide
BMC Structural Biology
Observation of glycine zipper and unanticipated occurrence of ambidextrous helices in the crystal structure of a chiral undecapeptide
Rudresh Acharya 2
Madhvi Gupta 0
Suryanarayanarao Ramakumar 1 2
Udupi A Ramagopal 3
Virander S Chauhan 0
0 Malaria Lab, International Centre for Genetic Engineering and Biotechnology , New Delhi , India
1 Bioinformatics Centre, Indian Institute of Science , Bangalore , India
2 Department of Physics, Indian Institute of Science , Bangalore , India
3 Department of Biochemistry, Albert Einstein College of Medicine , 1200, Morris Park Avenue, BRONX, New York 10461 , USA
Background: The de novo design of peptides and proteins has recently surfaced as an approach for investigating protein structure and function. This approach vitally tests our knowledge of protein folding and function, while also laying the groundwork for the fabrication of proteins with properties not precedented in nature. The success of these studies relies heavily on the ability to design relatively short peptides that can espouse stable secondary structures. To this end, substitution with α, β-dehydroamino acids, especially α, β-dehydrophenylalanine (∆Phe) comes in use for spawning well-defined structural motifs. Introduction of ∆Phe induces β-bends in small and 310-helices in longer peptide sequences. Results: The present report is an investigation of the effect of incorporating two glycines in the middle of a ∆Phe containing undecapeptide. A de novo designed undecapeptide, Ac-Gly1-Ala2-∆Phe3-Leu4-Gly5∆Phe6-Leu7-Gly8-∆Phe9-Ala10-Gly11-NH2, was synthesized and characterized using X-ray diffraction and Circular Dichroism spectroscopic methods. Crystallographic studies suggest that, despite the presence of L-amino acid (L-Ala and L-Leu) residues in the middle of the sequence, the peptide adopts a 310-helical conformation of ambidextrous screw sense, one of them a left-handed (A) and the other a right-handed (B) 310-helix with A and B being antiparallel to each other. However, CD studies reveal that the undecapeptide exclusively adopts a right-handed 310-helical conformation. In the crystal packing, three different interhelical interfaces, Leu-Leu, Gly-Gly and ∆Phe-∆Phe are observed between the helices A and B. A network of C-H...O hydrogen bonds are observed at ∆Phe-∆Phe and Gly-Gly interhelical interfaces. An important feature observed is the occurrence of glycine zipper motif at Gly-Gly interface. At this interface, the geometric pattern of interhelical interactions seems to resemble those observed between helices in transmembrane (TM) proteins. Conclusion: The present design strategy can thus be exploited in future work on de novo design of helical bundles of higher order and compaction utilizing ∆Phe residues along with GXXG motif.
De novo protein design endeavors to construct novel
polypeptide sequences that fold into well-defined
secondary and tertiary structures resembling those found in
native proteins. Many de novo design strategies have relied
on the known penchants of protein amino acids to
espouse various secondary structures leading to several
remarkable achievements [
]. Alternatively, the
amalgamation of conformationally restricted, non-protein
amino acids by chemical synthesis has led to triumphant
designs of secondary and super secondary structures that
mimic proteins [
]. In this regard, the ability of α,
βdehydrophenylalanine (∆Phe) to induce β-bends in small
and 310-helices in longer peptide sequences has been well
]. The presence of dehydroresidues in
peptides confers altered bioactivity as well as increased
resistance to enzymatic degradation . Recently designed
super secondary structures such as ∆Phe zippers and
helical hairpins highlight the potential of ∆Phe to introduce
long-range interactions in peptides and it has been noted
that the geometry of a 310-helix brings ∆Phe residues at i
and i+3 position into a stacking arrangement and the
structurally planar ∆Phe side-chains interdigitate to assist
the cooperative recognition of helices [
proteins, there is a wide interplay of weak non-covalent
interactions between secondary structural elements, to achieve
stability and overall compaction. In this context, in
transmembrane proteins it is observed that glycine residues
promotes close approach of helices, which permits not
only favourable vander Waals interactions of surrounding
side chains, but also in many cases, encourage interhelical
Cα-H...O hydrogen bonds [
]. Interestingly, it has
been found that the GXXXG motif elicits a level of
selfassociation in putative transmembrane helices and the
three-residue spacing between both glycines proves to be
optimal for the interaction. In an attempt to mimic
similar interactions and geometric features, we designed and
synthesized an undecapeptide,
structural features were characterized using X-ray diffraction
and Circular Dichroism spectroscopy. ∆Phe residues and
glycine residues as GXXG motif were at a two-residue
spacer to give rise to a 310-helical conformation. Thus the
peptide incorporates two GXXG like motif
(Gly5-∆Phe6Leu7-Gly8 and Gly8-∆Phe9 Ala10-Gly11) motif in the helix
region and one GXXXG (Gly1-Ala2-∆Phe3-Leu4-Gly5)
motif near the N-terminus. The bulky leucine residues
were placed in middle of the helical segment to ensure
that the peptide folds into a right-handed screw sense. A
310-helical conformation of ambidextrous screw sense is
established by X-ray diffraction. However, CD studies
reveal that a right-handed 310-helical conformation
dominate in solution. The preponderance of the right-handed
310-helical conformer is also confirmed using energy
calculation studies [Additional file 1]. An unanticipated
observation of ambidexterity of the peptide helices in the
crystal structure demonstrates the influence of global
interactions on the coexistence of left and right-handed
helices in the crystal structure. This is a novel observation
of a 310-helical dehydroundecapeptide mimicking
interhelical interactions as seen amongst transmembrane
Results and Discussion
The crystallographic details of the peptide are given in
(Table 1). Crystallographic studies suggest that, despite
the presence of L-Ala and L-Leu residues in the sequence,
the peptide has folded into two conformers in the crystal
lattice, conformer A and conformer B (Figure 1). From the
main chain conformation angles (Table 2) and the pattern
of intramolecular hydrogen bonds (Table 3), it is clear
that both right-handed as well as left-handed 310-helices
are present in the crystal structure. The average (ϕ,ψ)
values for 310- helical stretch (Ala2-Ala10) in conformer A are
(54°, 24°), whereas the average (ϕ,ψ) values for this
310helical stretch in conformer B are (-59°, -17°). The helices
are stabilized by intrahelical 4→1 hydrogen bonds (Table
3). Interestingly the four (L) amino acid residues, Ala2,
Leu4, Leu5 and Ala10 have taken the positive ϕ and ψ
values corresponding to the left-handed 310-helical
SuFtniegdrueearoceav1pieewptifdoer the molecular conformation of the
Stereo view for the molecular conformation of the
undeacapeptide. A denotes a left-handed 310-helix and B
aright-handed 310 helix. The helices A and B are antiparallel
to each other.
mation in conformer A (Table 2). In 310-helices, every
third residue would lie on the same face of the helix.
Consequently the side chains of the three ∆Phe residues in the
undecapeptide, ∆Phe3, ∆Phe6, and ∆Phe9 are stacked on
one face of the helix, residues Leu4, Leu7 and Ala10 lie on
second face of the helix, while Ala2, Gly5 and Gly8 lie on
third face of the helix. This arrangement of side chains
creates a column of protuberant side chains at 120° to each
other, resulting in the formation of grooves and wedges.
The two helices A and B are antiparallel to each other. The
angle between the two helical axes is 179°. It is observed
that in crystal lattice the helix A is surrounded by three B
helices, similarly helix B is surrounded by three A helices
forming ∆Phe-∆Phe, Leu-Leu and Gly-Gly helical
interfaces (Figure 2). The closest approach Cα-Cα distances
between the helices A and B at three interfaces was
observed to be different; 5.9Å at the ∆Phe-∆Phe interface,
3.9Å at the Gly-Gly interface and 5.4 Å at the Leu-Leu
FAirgruanrgee2ment of helices in crystal packing
Arrangement of helices in crystal packing. The figure
shows the arrangement of helices as viewed down the helical
axes. There are three interhelical interfaces viz. Gly-Gly (1),
∆Phe-∆Phe (2) and Leu-Leu (3).
interface (calculated using computer program Helixang
from CCP4 suite). Despite the closest approach of helices
at the Gly-Gly interface as compared to Leu-Leu interface,
energy calculation studies suggest that the Leu-Leu
interface has the maximum stability followed by Gly-Gly and
then ∆Phe-∆Phe interface (Additional file 1). In the crystal
lattice, the helices of similar handedness related by
translation symmetry are observed as approximate helical rods
aligned along z-axis. It is interesting to note that helices of
same handedness pack one above the other and stabilize
through head-to-tail kind of N-H...O hydrogen bonds;
N2...O10', and N12...O1, while the tail to tail hydrogen
bonding N12 (A)...O9' (B) is observed between the
helices of opposite handedness [
] (Table 4). A notable
feature in the crystal structure is that the two shape
compliment helices A and B are interacting through
extensive network of hydrogen bonds. At the Leu-Leu interface,
helices A and B are involved in N-H...O hydrogen bond
(Table 4). At the Gly-Gly interface the two conformers A
and B are held together by five Cα-H...O hydrogen bonds
all along the helical axis [
]. These backbone (Cα-H) to
backbone (carbonyl) hydrogen bonds observed between
Cα(Ala2), Cα(Gly5), and Cα(Gly8) of conformer A to O8',
O5' and O2' of conformer B respectively, and conversely
Cα(Gly5) and Cα(Gly8) of Conformer B to O5' and O2' of
conformer A respectively (Table 4), involve GXXG motifs
from the two helices (Fig. 3a, Table 4). At the ∆Phe-∆Phe
interface, helices A and B are held together by
symmetrically placed aromatic-backbone C-H...O hydrogen bonds
distributed all along the helical axis [
]. Hence C-H
(Phenyl)...O (carbonyl) hydrogen bonds are observed
between Cδ2 (∆Phe3), Cδ2 (∆Phe6) and Cδ2 (∆Phe9) of
conformer A to O6', O3' and O1 of conformer B
correspondingly. Similarly C-H (Phenyl)...O (carbonyl)
hydrogen bonds are observed between Cδ2 (∆Phe3), Cδ2
(∆Phe6) and Cδ2 (∆Phe9) of conformer B to O6', O3' and
O1 of conformer A respectively (Fig. 3b, Table 4). The
coexistence of right and left-handed helices favored by the
involvement of interhelical hydrogen bonds in the solid
state may be presumably to optimize helix-helix
interactions, suggesting that tertiary (global) interactions,
including overall vander Waals, hydrophobic,
FNiegtuwroerk3 of C-H...O hydrogen bonds at different interfaces
Network of C-H...O hydrogen bonds at different
interfaces. a) Stereo view for the network of Cα-H...O
hydrogen bonds at Gly-Gly interface. The GXXG motif has
promoted the close approach of opposite handed 310-helices
there by encouraging the vander Waals and Cα-H...O
interactions. b) Stereo view for the network of C-H...O hydrogen
bonds at ∆Phe-∆Phe interface.
static and hydrogen bond interactions can significantly
influence even the local secondary structural features that
involves amino acid residues close to each other in a
peptide sequence. Glycine residues (Gly5, Gly7) here seems to
act as surrogate D-amino acids by assuming left-handed
helical conformation [
]. In particular, the interaction
motif which involves the occurrence of aromatic C-H..O
hydrogen bonds and intercalation of aromatic side chains
between adjacent and antiparallel 310-helices of opposite
handedness is observed in other ∆Phe containing peptide
crystal structures analyzed earlier in our laboratory [
It seems that the two opposite handed helices in the
crystal packing seen have utilized a similar interaction motif
leading to their association with each other. Despite the
presence of opposite handed helices, the present peptide
is found to engage itself in extensive C-H...O hydrogen
bonds. A remarkable feature of the present peptide is the
observation of zipper like arrangement of multiple
CαH...O hydrogen bonds consistently at three residue
intervals at Gly-Gly interface, which may be termed as glycine
zipper. The distance of 3.9Å between the adjacent helices
at the Gly-Gly interface promotes packing interactions
between the helices. This similar geometry for interhelical
interaction is reportedly observed in transmembrane
helical proteins between helices involving GXXXG like
motifs. Although the four-residue spacing is strongly
preferred over other possible Gly patterns, reinforcing the
significance of the GXXXGXXXG sequence pattern.
Nevertheless, other spacings could lead to glycine zipper
packing if the Gly residues are placed on the same face of
the helix. Thus, the glycine zipper face may act as a magnet
for helix packing.
Circular Dichroism studies
The peptide has three ∆Phe residues interspersed by two
amino acid residues. The CD spectra display a negative
couplet (-, +) in acetonitrile, chloroform and
trifluoroethanol. A negative band is observed at about 295 nm and an
intense positive band at about 265 nm, with a crossover
point at ~280 nm (Figure 4). This CD pattern corresponds
to the absorption maximum at 270–280 nm and arises
from the dipole-dipole interactions between the charge
transfer electronic moments of the two dehydroamino
acid chromophores placed in a mutual fixed disposition
within the molecule. This pattern as reported earlier, is
typical of a right-handed 310-helix [
]. The varying
intensity of bands in different solvents suggests different
content of the 310-helical conformer. In methanol, the
spectrum shows a positive band at about 280 nm. This
could be possible when the styryl side chains of
dehydroresidues are placed on the opposite sides of the helix.
In this arrangement, no exciton splitting will be observed,
and the positive band at 280 nm arises from the
contributions of the noninteracting but chirally perturbed
chromophores. The very low intensity of bands in the CD
spectrum in methanol may be attributed to the polarity of
the solvent. It is known that folded peptide structures with
stabilizing hydrogen bonds are more stable in apolar
solvents than in polar ones. The peptide is found to
preferentially form a right-handed 310-helical conformer. The
difference between X-ray and CD interpretation may arise
due to conformational heterogeneity in the solid state that
can lead to crystallization of a minor conformer, driven by
favorable packing interactions. On the other hand, the
solution studies largely monitor the major species present
in solution. The stabilization of right-handed conformer
FCiDgusrpeec4trum in different solvents CD spectrum in different solvents.
over the left-handed 310-helical conformer is also
confirmed using energy calculation studies (Additional file
1). The CHCl3-MeOH titrations revealed a surprising but
interesting observation. At a concentration of 50:50
(chloroform: methanol), not only the right-handed 310-helical
structure is observed but there is also a steep rise in the
molar ellipticity value (Figure 5). It is possible that an
equal mixture of a polar (methanol) and an apolar
(chloroform) solvent provided some kind of amphiphilic
environment to the peptide, leading to enhanced stabilization
of the structure as compared to that in chloroform alone.
Following the above observation, the experiments were
performed in different lipomimetic solvents such as
aqueous SDS and aqueous TFE mixture. CD spectra of the
undecapeptide in SDS and TFE/water solution show
intense exciton-coupled band, characteristic of a
righthanded 310-helical conformer. Though the peptide was
completely insoluble in water but it was soluble in
different percentages of SDS/water and TFE/water (Figure 6a).
Thus the peptide is found to attain more stability in a
membranous environment. The band intensity in TFE/
water (40–70%) decreased with the decrease in the
percentage of TFE (Figure 6b) and increase in the water
content, which is deleterious for dehydrophenylalanine
containing structured peptides. However the decrease in
band intensity does not reflect in any conformational
change of the present peptide even at 40% TFE/water,
suggesting the overall stability of the peptide in a
membranous environment, provided by TFE/water mixture.
aFCtihg5luo0r:oe5f0o5rCmH-mCle3t:hManeoOl Htitration depicting maximum intensity
Chloroform-methanol titration depicting maximum intensity
at 50: 50 CHCl3: MeOH.
Variable temperature studies in 40% TFE/water show
maximum stability at 10°C, suggesting the effect of
lowering the temperature on the stability of the structure
(Figure 7). The explanation for the above observation could
be a result of TFE reinforcing hydrogen bonds between
carbonyl and amidic NH groups by the removal of water
molecules in the proximity of the solute and lowering the
dielectric constant of the surrounding milieu [
Thus the peptide attains more stability in membrane
mimetics at relatively low percentage, suggesting the
propensity of the peptide to exist in an ordered 310-helical
conformation in a hydrophobic environment and
depicting stabilization achieved by molecular association .
The present peptide,
Ac-Gly-Ala-∆Phe-Leu-Gly-∆Phe-LeuGly-∆Phe-Ala-Gly-NH2, provides the first example of
stability and compaction in interacting helices when glycine
residues are incorporated in the middle of the peptide
sequence. The incorporation of glycines in the form of
GXXG motif along with ∆Phe residue at two-residue
spacer has helped in maintaining the 310-helical
conformation in both solid as well as solution state. The
amalgamation of GxxG motif has not only facilitated the helices
to come close at the Gly-Gly interhelical interface but also
promoted the formation of glyzine zipper, where a zipper
like arrangement of Cα-H...O hydrogen bonds is observed.
The occurrence of weak C-H...O hydrogen bonds at
∆Phe∆Phe interface along with occurrence of main chain to
FCiDgusrpeec6tra in different lipomimetic solvents
CD spectra in different lipomimetic solvents. (a)
Different concentrations of SDS-water. (b) Different percentage
of aqueous TFE.
main chain Cα-H...O hydrogen bonds consistently at
three residue intervals at Gly-Gly helical interface
involving GXXG motifs seems to impart molecular association
and stabilization to the interacting helices. The
phenomenon of molecular association leading to stabilization of
the 310-helical conformer is also confirmed by the
solution state study. The present design can encourage the
peptide designers in pursuing the ambitious goal of de
novo design of helical bundles of higher order and
compaction utilizing ∆Phe residues along with GXXG motifs.
VT-CD spectrum. CD spectra in 40% TFE/water as a function of different temperatures.
Fmoc-protected amino acids for solid-phase peptide
synthesis were obtained from Novabiochem. The
undecapeptide was synthesized manually at a 0.5 mmol scale.
FmocRinkamide MBHA resin (Novabiochem) (0.5 mmol/g)
was used to afford carboxyl-terminal primary amide.
Couplings were performed by using carbodiimide. The ∆Phe
residue was introduced by dehydration of
Fmoc-aa-DLthreo-β-Phenyl Serine (AA = glycine or alanine) using
fused sodium acetate and freshly distilled acetic
anhydride as reported earlier [
]. All reactions were
monitored by TLC on precoated silica plates in 9:1
CHCl3MeOH system. The physical characterization of the
dipeptide synthons is given as follows: Fmoc-Gly-DL-Phe
(βOH)-OH: Yield = 91.4%, m.p. = 72–74°C, Rf = 0.40,
Fmoc-Gly-∆Phe-Azlactone: 93%, 102–104°C, 0.95,
Fmoc-Ala-DL-Phe (β-OH)-OH: 90%, 112–115°C, 0.3,
Fmoc-Ala-∆Phe-Azlactone: 91%, 142–145°C, 0.7. All the
couplings were followed by a five-minute reaction with
acetic anhydride and HOBT in DMF/DCM to cap any
unreacted amines. Fmoc deprotection was performed
with piperidine (20% in DMF). After addition of the final
residue, the amino terminus was acetyl-capped and the
resin was rinsed with DMF/DCM/MeOH and dried. The
final peptide deprotection and cleavage from the resin was
achieved with 10 ml of 95:2.5:2.5 TFA: H2O:
triisopropylsilane for two hours. The crude peptide was precipitated
with cold ether, lyophilized and purified by preparative
reverse phase HPLC. The crude peptide was purified by
RP-HPLC using water-acetonitrile gradient on Waters
Deltapak C18 (19 mm × 300 mm). A linear gradient of
acetonitrile from 10% to 70% over 60 mins at a flow rate of
6 ml/min was employed. The purified fractions were
pooled, lyophilized and stored at -20°C as dry powder.
RP-HPLC spectrum of the peptide is given (Figure 8).
Retention time: 41.5 mins. Peptide identity was
confirmed by mass spectrometer, C55 H70 N12 O12, calculated
mass 1091da, observed mass 1114 Da (sodium peak),
melting point: 160–165°C.
The peptide crystals were grown by the slow evaporation
of peptide solution (1:1 v/v) in ethanol and acetone
mixture. The X-ray diffraction data was collected using a
suitable crystal cryo cooled to 100 K in synchrotron radiation
source, at beam line X9A, Brookhaven National
Laboratory. The structure was solved by direct method using
SHELXS and was refined using full matrix least square
refinement employed in SHELXL [
]. The hydrogen
atoms were fixed using stereochemical criteria and were
allowed to ride on parent atoms. The crystallographic data
of the present peptide is deposited in CCDC
RFiPgHuPrLeC8of the peptide
RPHPLC of the peptide.
Circular Dichroism studies
CD spectra were recorded on a JASCO J-720 CD
spectropolarimeter. The spectra were acquired between 220–
330 nm (0.1 cm cell, peptide concentration ~100 µM) at
0.1 nm intervals with a time constant of 4 seconds and a
scan speed of 200 nm/min and averaged over 6 separate
scans. The spectra obtained were baseline corrected and
smoothed. Peptide concentration was determined using
the molar extinction coefficient of ∆Phe (~19,000
M-1cm1). CHCl3-methanol titration was carried out. CD spectra
were recorded at different concentrations of SDS and also
at different percentage of TFE/water. The CD spectra were
recorded in 40% TFE/water at variable temperatures.
The energy minimization for the present peptide was
performed using the SYBYL software package (version 7.0)
(1). The force field used was AMBER7 FF99 implemented
in SYBYL. The convergence criterion of 0.05 kcal/mol (Å)
as well as the non-bonded cut-off distance was set to 8Å.
The partial charges on protein residues were AMBER7 F99
all-atom charges. A value of 1 was set out for dielectric
constant for these peptides. The details of energy
calculation values are given as additional file 2.
List of Abbreviations
DMF: N, N-Dimethylformamide
Rinkamide MBHA resin:
SDS: Sodium dodecyl sulphate
TFA: Trifluoroacetic acid
TLC: Thin layer chromatography
RA solved the crystal structure of the peptide, carried out
energy calculation studies, analysis and interpretation of
crystal data. MG carried out the peptide synthesis,
purification and characterization, acquired the CD spectra and
performed the analysis of the CD data. UAR collected and
processed the synchrotron diffraction data for the crystal.
RA, MG, SR, and VSC conceived of the study, and
participated in its design and coordination and helped to draft
the manuscript. All authors read and approved the final
Additional file 1
Energy Calculation Studies. Energy values at various interfaces, calculated
using software SYBYL.
Click here for file
Additional file 2
Energy Calculation Studies.
Click here for file
The financial support from the Department of Science and Technology,
India is acknowledged. The financial support from WHO, is also
acknowledged. We would like to thank Prof. Faizan Ahmad at Jamia Millia Islamia
for his consent in using the Circular Dichroism facility in his laboratory. We
thank department of Biotechnology, India for access to facilities at
Bioinformatics and interactive graphics facility, I.I.Sc, Bangalore. RA would like to
thank IBM-CAS fellowship.
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