Noncovalent multivalent assembly of jun peptides on a leucine zipper dendrimer displaying fos peptides

Article abstract of DOI:10.1021/ol0485262

(Chemical Equation Presented) The synthesis and characterization of a new leucine-zipper dendrimer (LZD) is reported that displays four copies of the peptide corresponding to the coiled-coiled dimerization domain of Fos. Circular dichroism spectroscopy, fluorescence titration, and sedimentation equilibrium experiments demonstrate that Fos-LZD can noncovalently assemble four copies of the peptide corresponding to the coiled-coil domain of Jun. This work provides the basis for the future construction of noncovalently assembled multivalent protein assemblies displaying any protein of interest.

Full text of DOI:10.1021/ol0485262

ORGANIC
LETTERS
2
004
Vol. 6, No. 20
561-3564
Noncovalent Multivalent Assembly of
Jun Peptides on a Leucine Zipper
Dendrimer Displaying Fos Peptides
3
Min Zhou and Indraneel Ghosh*
Department of Chemistry, UniVersity of Arizona, Tucson, Arizona 85721
ghosh@email.arizona.edu
Received July 27, 2004
ABSTRACT
The synthesis and characterization of a new leucine-zipper dendrimer (LZD) is reported that displays four copies of the peptide corresponding
to the coiled-coiled dimerization domain of Fos. Circular dichroism spectroscopy, fluorescence titration, and sedimentation equilibrium experiments
demonstrate that Fos-LZD can noncovalently assemble four copies of the peptide corresponding to the coiled-coil domain of Jun. This work
provides the basis for the future construction of noncovalently assembled multivalent protein assemblies displaying any protein of interest.
7
There is much interest in utilizing noncovalent interactions
to control the biomimetic assembly of unimolecular, multi-
meric, and polymeric structures for biological and nanotech-
assembly utilizing either hydrogen bonding or metal coor-
8
dination. We have recently combined aspects of peptide self-
assembly and dendrimeric architecture in the synthesis of
leucine zippers appended to a core dendrimer, providing the
necessary framework for constructing multivalent leucine
zipper dendrimers (LZDs). These LZDs can noncovalently
assemble cognate helical peptides at their periphery. The
ability to design biomimetic modular multivalent systems is
of much interest in targeting cellular receptors, DNA, and
in assembling novel architectures.
We have focused on leucine zippers or, more generically,
helical coiled-coils as our self-assembly unit as they are
very amenable to design and many orthogonal pairs exist
1
nological applications. Many elegant unimolecular, confor-
mationally unique structures have been recently designed
including â-peptides2 and template-assembled synthetic
9
3
proteins (TASPs) and other novel foldamers. At the same
time more complex polymeric self-assembling structures
have also been designed, which include cyclic-peptide
10
11
1
,3
4
5
6
nanotubes, peptide-hydrogels, and even PNA-microgels.
Dendrimer construction has also witnessed the use of self-
1
2
1
3
(
1) Lindsey, J. S. New J. Chem. 1991, 15, 153-180.
(2) Appella, D. H.; Christianson, L. A.; Klein, D. A.; Powell, D. R.;
Huang, X.; Barchi, J. J., Jr.; Gellman, S. H. Nature 1997, 387, 381-384.
(7) Zimmerman, S. C.; Zeng, F. W.; Reichert, D. E. C.; Kolotuchin, S.
V. Science 1996, 271, 1095-1098.
(8) Kawa, M.; Frechet, J. M. J. Chem. Mater. 1998, 10, 286-296
(9) Zhou, M.; Bentley, D.; Ghosh, I. J. Am. Chem. Soc. 2004, 126, 734-
735.
(
3) (a) Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S.
Chem. ReV. 2001, 101, 3893-4011. (b) Mutter, M.; Vuilleumier, S. Angew.
Chem., Int. Ed. Engl. 1989, 28, 535-554.
(4) Ghadiri, M. R.; Granja, J. R.; Milligan, R. A.; McRee, D. E.;
Khazanovich, N. Nature 1993, 366, 324-327.
(10) (a) Mammen, M.; Choi, S. K.; Whitesides, G. M. Angew. Chem.,
Int. Ed. 1998, 37, 2755-2794. (b) Kiessling, L. L.; Gestwicki, J. E.; Strong,
L. E. Curr. Opin. Chem. Biol. 2000, 4, 696-703. (c) Gestwicki, J. E.; Cairo,
C. W.; Strong, L. E.; Oetjen, K. A.; Kiessling, L. L. J. Am. Chem. Soc.
2002, 124, 14922-14933.
(11) Kim, J. S.; Pabo, C. O. Proc. Natl. Acad. Sci. U.S.A. 1998, 95,
2812-2817.
(
5) (a) Petka, W. A.; Harden, J. L.; McGrath, K. P.; Wirtz, D.; Tirrell,
D. A. Science 1998, 281, 389-392. (b) Schneider, J. P.; Pochan, D. J.;
Ozbas, B.; Rajagopal, K.; Pakstis, L.; Kretsinger, J. J. Am. Chem. Soc. 2002,
1
24, 15030-15037.
6) Cao, R.; Gu, Z. Y.; Hsu, L.; Patterson, G. D.; Armitage, B. A. J.
Am. Chem. Soc. 2003, 125, 10250-10256.
(
1
0.1021/ol0485262 CCC: $27.50
© 2004 American Chemical Society
Published on Web 09/09/2004

naturally14 or can be selected.15 This potentially allows for
the construction of numerous orthogonal self-assembling
systems, which is not possible with currently utilized
streptavidin-biotin6 or Ni-chelation based multivalent
assembly systems. Helical coiled-coils organize their amino
acids in a repeating heptad pattern, which when numbered
a-g places a specific hydrophobic residue, such as leucine,
at the a and d positions to create the knob-hole like
hydrophobic interface.
orthogonal to a multitude of other natural coiled-coil pairs14
and provides a rational starting point for the future assembly
of multiple mutually exclusive LZD/cargo pairs. Multiple
designed leucine zippers, as utilized in our previous design,
have yet to be systematically tested for orthogonality in
competitive cellular environments.
,16
17
In our design, peptides corresponding to the coiled-coil
domains of the well-characterized bZIP protein Fos and
18,19
Jun
were chosen as starting points as our self-assembling
Our previous design strategy utilized electrostatically
driven complexation of a coiled-coil tetramer upon the
generation-0 PAMAM dendrimeric core at either low or high
pH regimes. To expand the utility of our approach toward
the facile assembly of discrete multivalent supramolecules
at neutral pH, herein we report the synthesis and character-
ization of a new LZD that is functional at neutral pH. This
new LZD displays the coiled-coil domain of the human
transcription factor, Fos, and assembles four copies of a
peptide corresponding to the coiled-coil domain of its cellular
binding partner Jun (Figure 1). Such a system can have great
units. We anticipated that the covalent attachment of four
coiled-coils to a dendrimer would result in the undesirable
intramolecular stabilization of coiled-coils, which is useful
3
in TASP assemblies. We reasoned that the less stable Fos
coiled-coil would be suitable for decorating the core den-
drimer on the basis of literature precedence that the Fos/Fos
homodimer is less stable than the Jun/Jun homodimer, both
of which are an order of magnitude less stable than the Fos/
1
8,19
Jun heterodimer.
The peptides corresponding to the coiled-coil domains of
Fos and Jun, PFos and PJun were synthesized by standard
solid-phase Fmoc chemistry on a Rink amide resin. Peptides
were cleaved and purified by HPLC and subsequently
characterized by amino acid analysis and MALDI mass
spectrometry (see Supporting Information). The maleimido-
functionalized generation-0 PAMAM dendrimer was reacted
chemoselectively with a unique Cys at the N-terminus of
0 4
the Fos peptide to generate D -Fos in 20-25% yield
(Supporting Information). We also synthesized the Jun
peptide (PJun), with an added 7-hydroxycoumarin (PJun-
HC) followed by a Gly-Gly linker at the N-terminus. PJun-
HC was synthesized to monitor the noncovalent assembly
0 4
of PFos/PJun-HC and D -Fos /4PJun-HC complexes utilizing
1
8
the Gellman assay, which monitors the relief of excimer-
mediated quenching of the PJun-HC homodimer. The
respective sequence of the peptides and LZD-dendrimer used
in this study are shown in Figure 1.
Our initial studies focused on characterization of D
0 4
-Fos
in comparison to PFos. We predicted that D -Fos would
0
4
Figure 1. PFos (red) is shown appended to the generation-0
PAMAM dendrimer (purple ball) to result in the LZD D -Fos .
0 4
The sequences of PFos (red), PJun (blue), and 7-hydroxycoumarin-
appended PJun (PJun-HC) are also shown.
fold into two coiled-coils (Figure 1) as a disulfide linked
Fos-peptide homodimer has been previously been shown to
be completely folded. We utilized circular dichroism (CD)
19
0 4
to monitor the secondary structure of the dendrimer D -Fos
in comparison to PFos at pH 7 with 10 mM phosphate, 100
mM NaCl, 1 mM DTT (buffer A). The CD spectra of PFos
utility in the noncovalent multivalent display of any protein
fused to a Jun-coiled-coil. This system is especially attractive
as the Fos/Jun coiled-coil pair has been shown to be
and the LZD, D
0
4
-Fos , were significantly different (Figure
2
a) and indicated that PFos is primarily unfolded at 25 °C,
whereas the D
tions. The greater helicity of D
0
-Fos
4
is 85% folded under the same condi-
-Fos is likely due to the
0
4
(
12) (a) Crick, F. H. C. Acta Crystallogr. 1953, 6, 689-697. (b) Lupas,
anticipated covalent stabilization as reported for the disulfide-
tethered Fos peptides and also observed in coiled-coil
A. Trends Biochem. Sci. 1996, 21, 375-382.
(
13) (a) Oshea, E. K.; Lumb, K. J.; Kim, P. S. Curr. Biol. 1993, 3, 658-
6
3
2
67. (b) Monera, O. D.; Kay, C. M.; Hodges, R. S. Biochemistry 1994, 33,
862-3871. (c) Bilgicer, B.; Fichera, A.; Kumar, K. J. Am. Chem. Soc.
001, 123, 4393-4399. (d) Schnarr, N. A.; Kennan, A. J. J. Am. Chem.
20
assembly in the seryl tRNA synthetase. To interrogate the
0 4
stability of D -Fos in comparison to PFos, we monitored
the temperature-dependent change in secondary structure
Soc. 2001, 123, 11081-11082.
(14) Newman, J. R. S.; Keating, A. E. Science 2003, 300, 2097-2101.
(15) (a) Arndt, K. M.; Pelletier, J. N.; Muller, K. M.; Alber, T.; Michnick,
S. W.; Pluckthun, A. J. Mol. Biol. 2000, 295, 627-639. (b) Kim, B. M.;
Oakley, M. G. J. Am. Chem. Soc. 2002, 124, 8237-8244. (c) Ghosh, I.;
Hamilton, A. D.; Regan, L. J. Am. Chem. Soc. 2000, 122, 5658-5659.
(18) Daugherty, D. L.; Gellman, S. H. J. Am. Chem. Soc. 1999, 121,
4325-4333.
(19) (a) O’Shea, E. K.; Rutkowski, R.; Stafford, W. F. D.; Kim, P. S.
Science 1989, 245, 646-648. (b) Kohler, J. J.; Schepartz, A. Biochemistry
2001, 40, 130-142.
(
16) Green, N. M. Method Enzymol. 1990, 184, 51-67.
(17) Griffith, B. R.; Allen, B. L.; Rapraeger, A. C.; Kiessling, L. L. J.
Am. Chem. Soc. 2004, 126, 1608-1609.
(20) Oakley, M. G.; Kim, P. S. Biochemistry 1997, 36, 2544-2549.
3562
Org. Lett., Vol. 6, No. 20, 2004

Figure 3. CD analysis of D
a) The uncorrected CD spectra of 2 µM D
µM PJun (purple circle), and 2 µM D -Fos
circle). The curve (black cross) represents the calculated weighted
average spectra for 2 µM D -Fos + 16 µM PJun. (b) Change in
PJun helicity upon complexation with Do-Fos at [θ]222 in the
presence of 2 µM D -Fos . Change in θ222 of PJun is calculated
from (θ222 [D -Fos ] All samples
were in buffer A.
0
-Fos
4
and PJun complex formation.
-Fos (green circle), 16
+ 16 µM PJun (blue
Figure 2. CD secondary structural analysis of PFos and D
0
-Fos
-Fos
green) at 25 °C in pH 7.0 buffer containing 10 mM phosphate,
00 mM NaCl, and 1mM DTT (buffer A). (b) Thermal unfolding
of 20 µM PFos and 5 µM D -Fos in buffer A followed by the
change in CD signal at 222 nm.
4
.
4
(
0
4
(
(
a) CD spectra of 20 µM PFos (red) and 5 µM dendrimer D
0
0
4
1
0
4
0
4
4
0
4
)
[D -Fos +PJun] - (θ222)[PJun] - (θ222)
0
4
0
4
(Figure 2b) by following the change at [θ]222 by CD
spectroscopy.
m
In the CD melting curve, the apparent T (where 50% of
the structures are folded) of D
on PFos concentration) is ∼40 °C higher than that of PFos
at 20 µM. The mean residue ellipticity at [θ]222 of D -Fos
at 4 and 96 °C was taken to represent the theoretical
maximum folded and unfolded baselines for D -Fos and
PFos at these concentrations. Given the relative stability of
-Fos we wanted to ensure that it did not form higher order
0
-Fos
4
at 5 µM (20 µM based
fully folded, and 16 µM PJun is primarily unfolded. If there
were no interaction between D -Fos and PJun, then the
experimental spectra of 2 µM D -Fos with 16 µM Jun would
be equivalent to the sum of the spectra for D -Fos (2µM)
and PJun (16 µM). However, the CD spectra clearly show
that 16 µM PJun + 2 µM of D -Fos is considerably more
0
4
0
4
0
4
0
4
0
4
0
4
D
0
4
helical than that for the calculated addition spectra. This
oligomers that would complicate analysis for PJun complex-
ation studies. Toward this end we measured the molecular
weight and oligomerization states of the PJun, PJun-HC, and
change in helicity can be directly attributed to change in PJun
secondary structure upon complexation with D -Fos . The
0 4
result of the complete CD titration experiments is shown in
Figure 3b, where the relative change in PJun helicity at 222
0 4
D -Fos utilizing sedimentation equilibrium (Supporting
Information). Measurements were made on an analytical
ultracentrifuge at 20 °C in PBS at three different concentra-
tions. We found that PJun and PJun-HC existed in equilib-
nm obtained from the subtraction spectra of (θ222
)
[D -Fos +PJun]
0 4
- (θ222)[PJun] - (θ222)
[D -Fos ] is shown as a function of
0
4
increasing PJun concentration. The titration curve plateaus
rium between monomer and dimer, whereas D
0
-Fos
4
existed
at 16 µM PJun, suggesting that an 8-fold excess (16 µM) of
only as a monomer. The dissociation constant of PJun was
found to be 45 µM, which was 3.5-fold weaker than that for
PJun-HC, which was found to be 13 µM.
Gellman and co-workers have previously reported a similar
increase in stability of a Jun-based peptide homodimer upon
the incorporation of 7-hydroxycoumarin.18 Having estab-
lished that PJun was only partially helical between 0 and 50
PJun is required for the complete complexation of 2 µM D
0
-
Fos , where all the PFos tethered to the LZD is completely
4
occupied by PJun. This analysis, although confirming our
design strategy, is complicated by the concentration-depend-
ent monomer-dimer equilibrium of PJun.
We further evaluated the complexation of D
0
-Fos
PJun utilizing the Gellman assay, where titration of PJun-
HC with either D -Fos or PFos is expected to lead to an
4
with
1
8
µM and that D
we predicted that it would be possible to monitor the
assembly of four copies of peptide Jun upon the LZD D
Fos utilizing CD spectroscopy. In our CD experiment, we
assumed that the overall helicity of PJun would increase as
it was complexed by D -Fos4. To test this, we made a series
of samples in pH 7.0 buffer (10 mM phosphate, 100 mM
NaCl, 1 mM DTT), such that the concentration of D -Fos
0
-Fos
4
was completely helical and monomeric,
0
4
increase in PJun-HC fluorescence due to an increase in the
average distance between the 7-hydroxycoumarins attached
to dimeric PJun-HC. The change in PJun-HC fluorescence
was monitored by excitation at 386 nm and emission at 447
nm. The concentration of PJun-HC was held constant at 1.67
µM, while the concentration of D -Fos (0 to 5 µM) or PFos
0 4
(0 to 20 µM) was varied. The results are shown in Figure
0
-
4
0
0
4
was constant at 2 µM (8 µM based on PFos concentration),
while the concentration of PJun was varied from 0 to 40
4a, where the change in PJun-HC fluorescence is plotted with
respect to the molar ratio of [PFos]/[PJun-HC], ([D
[PJun-HC]. The results from the PJun-HC titration clearly
show that both D -Fos and PFos can complex PJun-HC,
0 4
-Fos ])/
µM in the presence and absence of D
0
-Fos
4
. The results for
-Fos + 16 µM
-Fos is
2
µM D -Fos , 16 µM PJun, and 2 µM of D
0
4
0
4
0
4
PJun is shown in Figure 3a. It is clear that 2 µM D
0
4
resulting in relief of excimer quenching as the 7-hydroxy-
Org. Lett., Vol. 6, No. 20, 2004
3563

data indicated that when the molar ratio of D
:4, the data was best fit by three species corresponding to
-Fos /2PJun, PJun-monomer and PJun dimer. However,
when the molar ratio of D -Fos /PJun was 1:8, the data was
best fit to three species corresponding to D -Fos /4PJun,
0 4
-Fos /PJun was
1
D
0
4
0
4
0
4
PJun-monomer, and PJun dimer (Supporting Information).
Thus, these sedimentation equilibrium results along with the
fluorescence and CD experiments indicate that we have
successfully assembled four copies of PJun on the D
scaffold to afford a noncovalent supramolecular complex of
0,000 Da in molecular weight. The complexationof PJun
with D -Fos could follow a two-step process, where first
two of the PFos peptides on D -Fos unfold and bind 2 equiv
0 4
-Fos
4
0
4
0
4
of PJun, following which the next two Fos peptides unfold
and bind another 2 equiv of PJun. Alternatively, the PJun
peptides could bind the Fos peptides appended to the
dendrimer core one at a time, which does not agree with
our sedimentation equilibrium data and would also be
energetically unfavorable.
This work demonstrates the noncovalently assembly of
four coiled-coil peptides upon a leucine zipper dendrimer-
displaying scaffold characterized by CD, fluorescence, and
sedimentation equilibrium experiments. These studies also
show that the significant covalent stabilization of leucine
zippers by the dendrimeric scaffold has to be overcome for
the successful noncovalent assembly of cognate peptides.
Future work in this area will entail the expression of proteins
directly attached to leucine zippers, such as PJun, such that
Figure 4. Fluorescence titration of PJun-HC. In (a) the titration
of PJun-HC by PFos (() and D -Fos (O) is shown. In (b) the same
data are shown, but D -Fos concentration is expressed as [D
Fos ]/4 (0) to compare more directly with PFos (() on a per leucine
zipper basis. PJun-HC was constant at 1.67 µM; excitation was at
86 nm, and emission recorded at 447 nm.
0
4
0
4
0
-
4
3
coumarins are no longer held in close proximity. Moreover,
in both cases recovery of fluorescence plateaus when the
concentration of PFos is over 6-fold that of PJun-HC.
However, whereas PFos was observed to recover 50% of
PJun-HC fluorescence at 0.7 molar equiv, D -Fos could only
0 4
recover 50% of PJun-HC fluorescence at 1.9 molar equiv
based on a per leucine zipper basis (Figure 4b). This 2.7-
fold difference in the ability of D -Fos versus PFos to
0 4
successfully complex PJun-HC can be attributed to the
energetic penalty for first having to unfold the covalently
0 4
they can be complexed upon LZDs, such as D -Fos , to
instantly afford functional multivalent protein complexes. The
orthogonality of different leucine-zipper pairs can potentially
allow for the coexistence of many such assemblies that can
stabilized Fos peptides dimers on D
ation with PJun-HC. Alternatively, it is possible that the
assembly of four PJun peptides on the D -Fos is sterically
0
-Fos
4
prior to complex-
11
be utilized for DNA binding, targeting receptors on cell-
10
surfaces, or in mimicking multivalent enzymatic com-
0
4
21
plexes.
demanding. It is also interesting to note that the absolute
recovered fluorescence of PJun-HC was 25% less when
Acknowledgment. We thank Tom Baldwin and Mike
Cusanovich for the use of their instruments. This work was
supported by the donors of the Petroleum Research Fund
for a PRF Type G grant and by the Research Corporation
for a Research Innovation Award.
titrated with D
due to the four 7-hydroxycoumarins still being held in close
proximity when decorated upon the D -Fos LZD scaffold
Supporting Information).
Sedimentation equilibrium was utilized to directly inter-
rogate the D -Fos /PJun complex formation. On the basis
0 4
-Fos relative to PFos, which is very likely
0
4
(
Supporting Information Available: Synthesis, charac-
terization, fluorescence and sedimentation equilibrium data
for D -Fos , PFos, PJun, and PJun-HC. This material is
0 4
available free of charge via the Internet at http://pubs.acs.org.
0
4
of our CD and fluorescence titration experiments we
estimated that an 8-fold excess of PJun was required to
complex all four sites on D
equilibrium experiments two different molar ratios were
utilized where the D -Fos /PJun was held at relative ratios
of either 1:4 or 1:8. Analysis of the sedimentation equilibrium
0 4
-Fos . Thus, in the sedimentation
OL0485262
0
4
(21) Kumar, P.; Li, Q.; Cane, D. E.; Khosla, C. J. Am. Chem. Soc. 2003,
125, 4097-4102.
3564
Org. Lett., Vol. 6, No. 20, 2004