Simultaneous directed assembly of three distinct heterodimeric coiled coils

Article abstract of DOI:10.1021/ol801461a

(Figure Presented) We describe simultaneous formation of three distinct heterodimeric coiled coils from a mixture of six different peptides. The choice among electrostatically viable complexes is governed by alignment of buried core residues, including a

Full text of DOI:10.1021/ol801461a

ORGANIC
LETTERS
2008
Vol. 10, No. 17
3797-3800
Simultaneous Directed Assembly of
Three Distinct Heterodimeric Coiled
Coils
Maria L. Diss and Alan J. Kennan*
Department of Chemistry, Colorado State UniVersity, Fort Collins, Colorado 80523
kennan@lamar.colostate.edu
Received June 30, 2008
ABSTRACT
We describe simultaneous formation of three distinct heterodimeric coiled coils from a mixture of six different peptides. The choice among
electrostatically viable complexes is governed by alignment of buried core residues, including a fundamentally new interaction that exploits
urea-terminated side chains. Buried urea/urea contacts lead to extremely stable dimeric coiled coils, with Tm values between 63 and 79 °C.
Core ureas can also form stable complexes with a variety of other polar groups, including guanidines, acids, and amides.
Programmed molecular self-assembly is a powerful route to
otherwise prohibitively complicated structures, consistent
with its extensive application in biological systems. Such
natural scaffolds provide building blocks for advanced
design.1 As components are simplified, strategies for selective
recognition become correspondingly more complicated. As
such, introduction of new specificity elements, particularly
those that complement rather than replace existing ones,
significantly enhances downstream design capabilities.
Fundamental studies have produced well-characterized
coiled coils of varying peptide number and orientation,
formed by homo- or heteromeric strand binding and exhibit-
ing a broad range of stabilities up to and exceeding those
found in natural systems.2 In turn, these basic efforts have
facilitated application in protein folding models, materials
chemistry, and biotechnology.3
This widespread utility underscores the value of new
methods for specific coiled coil construction. We have
recently deveolped new motifs derived from a well-known
Among nature-inspired recognition architectures, design
and application of alpha helical coiled coils has drawn
considerable attention. Such systems bundle supercoiled
helical peptides, driven by hydrophobic packing of side
chains alternately spaced by 3 or 4 residues. This pattern
divides the sequence into seven-residue heptads (a-g) in
which hydrophobic a/d side chains are often flanked by polar
e/g residues that establish interhelical polar contacts.
(2) Coiled-coil reviews: (a) Woolfson, D. N. AdV. Protein Chem. 2005,
70, 79–112. (b) Parry, D. A. D.; Squire, J. M. AdV. Protein Chem. 2005,
70, 1–10. (c) Lupas, A. N.; Gruber, M. AdV. Protein Chem. 2005, 70, 37–
78. (d) Cheng, R. P. Curr. Opin. Struct. Biol. 2004, 14, 512–520.
(3) (a) Dong, H.; Hartgerink, J. D. Biomacromolecules 2007, 8, 617–
623. (b) Kammerer, R. A.; Steinmetz, M. O. J. Struct. Biol. 2006, 155,
146–153. (c) Papapostolou, D.; Smith, A. M.; Atkins, E. D. T.; Oliver,
S. J.; Ryadnov, M. G.; Serpell, L. C.; Woolfson, D. N. Proc. Natl. Acad.
Sci. U.S.A. 2007, 104, 10853–10858. (d) Zimenkov, Y.; Dublin, S. N.; Ni,
R.; Tu, R. S.; Breedveld, V.; Apkarian, R. P.; Conticello, V. P. J. Am.
Chem. Soc. 2006, 128, 6770–6771. (e) Shen, W.; Zhang, K.; Kornfield,
J. A.; Tirrell, D. A. Nat. Mater. 2006, 5, 153–158. (f) Yuzawa, S.; Mizuno,
T.; Tanaka, T. Chem.-Eur. J. 2006, 12, 7345–7352. (g) Woolley, G. A.;
Jaikaran, A. S. I.; Berezovski, M.; Calarco, J. P.; Krylov, S. N.; Smart,
O. S.; Kumita, J. R. Biochemistry 2006, 45, 6075–6084.
(1) (a) Goodman, C. M.; Choi, S.; Shandler, S.; DeGrado, W. F. Nat.
Chem. Biol. 2007, 3, 252–262. (b) Hill, D. J.; Mio, M. J.; Prince, R. B.;
Hughes, T. S.; Moore, J. S. Chem. ReV. 2001, 101, 3893–4011. (c) Gellman,
S. H. Acc. Chem. Res. 1998, 31, 173–180.
10.1021/ol801461a CCC: $40.75
Published on Web 08/12/2008
2008 American Chemical Society

basic strategy: alignment of buried polar groups. A single
polar side chain in a core a or d position of each
component peptide reliably favors complexes juxtaposing
those groups.4 In this vein, we have recently demonstrated
that variable-length guanidinylated core residues support
formation of well-defined dimeric coiled coils with a
variety of binding partners.5 In particular, guanidine (Gdn)/
aspartate pairs proved compatible with dimer formation
in the presence of alternate partners bearing core aspar-
agines, adding a significant dimension to possible as-
sembly systems. Given this success, we sought to discover
additional viable recognition partners and in particular
focused on neutral pairs that might emulate the strong
thermal stability of Asn/Asn contacts.
to discourage homodimer formation. Interactions with natural
polar residues and chain-shortened Arg derivatives were
measured using some of our previously reported sequences.5
Peptides were prepared via standard solid-phase methods,
augmented by our methods for on-resin urea formation during
solid-phase peptide synthesis.8
Our determination of urea-core feasibility began with
evaluation of urea/urea contacts. By design, each acidic
peptide (Glu at e/g) can pair only with one of the three basic
ones (Lys at e/g), affording nine possible mixtures. Equimolar
solutions of each combination examined by circular dichro-
ism (CD) spectroscopy exhibited wavelength and thermal
denaturation profiles consistent with coiled coil formation
(Figure 2).9 Significantly, spectra for 1:1 mixtures exhibited
Urea/urea recognition has a vast portfolio of applications
in synthetic self-assembly systems. Here we report ap-
plication of buried urea recognition in heterodimeric coiled
coil formation, with thermal stabilities up to and even
slightly exceeding that of the corresponding Asn/Asn
system (Tm ) 51-79 °C vs 77 °C for the Asn pair). Most
strikingly, use of a citrulline pair allows simultaneous
formation of three distinct heterodimers from a six-peptide
mixture, a problem of exponentially higher complexity
than even the already-difficult four-component system
previously reported.
Peptide sequences for probing core urea interactions were
derived from the Acid-p1/Base-p1 heterodimer reported by
Kim and co-workers, itself inspired by the coiled-coil
domains of natural proteins (Fos, Jun, GCN4).7 Each peptide
contains a core urea side chain at position 14, spaced from
the backbone by one (pUr), two (pUr*), or three (pCit)
methylenes (Figure 1). Acidic and basic derivatives of each
Figure 2. Coiled coils with buried urea side chains. Thermal
denaturation CD curves for the indicated equimolar peptide mixtures
with identical (A) or distinct (B) urea side chains at a core a
position. In (B), closed/open circles of the same color denote
complexes that differ by exchange of polar residue between acidic
and basic sequences. The Asn/Asn dimer is given in (A) for
comparison.6
room-temperature helicities and thermal stabilities well in
excess of calculated component weighted average signals,
consistent with complex formation.10 Analytical ultracen-
trifugation supports dimer formation in all three like-group
pairs, although the pUrK/pUrE complex shows some evidence
of higher-order behavior.10
(4) (a) Straussman, R.; Ben-Ya’acov, A.; Woolfson, D. N.; Ravid, S. J.
Mol. Biol. 2007, 366, 1232–1242. (b) Lear, J. D.; Gratkowski, H.; Adamian,
L.; Liang, J.; DeGrado, W. F. Biochemistry 2003, 42, 6400–6407. (c)
Schneider, J. P.; Kretsinger, J. J. Am. Chem. Soc. 2003, 125, 7907. (d)
McClain, D. L.; Gurnon, D. G.; Oakley, M. G. J. Mol. Biol. 2002, 324,
257–270. (e) Akey, D. L.; Malashkevich, V. N.; Kim, P. S. Biochemistry
2001, 40, 6352–6360. (f) Oakley, M. G.; Kim, P. S. Biochemistry 1998,
37, 12603–12610. (g) Zeng, X.; Herndon, A. M.; Hu, J. C. Proc. Natl.
Acad. Sci. U.S.A. 1997, 94, 3673–3678. (h) Schneider, J. P.; Lear, J. D.;
DeGrado, W. F. J. Am. Chem. Soc. 1997, 119, 5742. (i) Gonzalez, L., Jr.;
Woolfson, D. N.; Alber, T. Nat. Struct. Biol. 1996, 3, 1011–1018. (j) Lumb,
K. J.; Kim, P. S. Biochemistry 1995, 34, 8642–8.
Figure 1. Peptides used. Helical wheel projection demonstrates
interactions in a heterodimer containing one acidic (pXaaE) and
one basic (pXaaK) peptide (general sequences below). Structures
of unnatural buried polar residues for Ur/Ur*/Gd/Gd* peptides are
as shown (position 14 in the sequence, denoted with a Z). Peptides
with natural core polar residues are also listed. Underlined lysine
side chains are capped with acetamidobenzoyl groups as spectro-
scopic labels.
(5) Diss, M. L.; Kennan, A. J. J. Am. Chem. Soc. 2008, 130, 1321–
1327.
(6) Solutions for all CD experiments contained 10 µM total peptide in
PBS buffer (150 mM NaCl, 10 mM phosphate, pH 7.0). See Supporting
Information for corresponding wavelength traces.
(7) O’Shea, E. K.; Lumb, K.; Kim, P. S. Curr. Biol. 1993, 3, 658–667.
(8) Diss, M. L.; Kennan, A. J. Biopolymers 2007, 86, 276–281.
(9) See Supporting Information for wavelength traces and plots of 1:1
mixtures vs individual components and weighted averages.
sequence were prepared (denoted with E or K subscripts,
respectively), in which all e/g positions contain Glu or Lys,
(10) See Supporting Information for further details.
3798
Org. Lett., Vol. 10, No. 17, 2008

In general, use of a neutral polar group had the desired
effect on heterodimer stability (Tm ) 63-79 °C, Table 1).
than the amide/urea ones. As with the guanidine core
residues, little to no preference was exhibited for binding to
either Asp or Glu partners.
Comparing the melting curves for each urea derivative
with its nonurea partners reveals some general trends (Figure
3). The short-chain guanidines were generally the weakest
Table 1. Observed Tm Values for Urea Core Pairs
sample
Tm (°C)
sample
Tm (°C)
pUrK/pUrE
pUr*K/pUr*E
pCitK/pCitE
pUrK/pUr*E
pUrE/pUr*K
63
63
79
63
65
pUrK/pCitE
pUr*K/pCitE
pUrE/pCitK
pUr*E/pCitK
63
67
71
75
In particular, the pCitK/pCitE sample displayed a Tm nomi-
nally higher than the parent pAsnK/pAsnE system (79 vs 77
°C), a significant improvement over our previously most
stable non-Asn core system (featuring a guanidine/carboxy-
late pair with Tm ) 67 °C).5 Any influence of polar residue
side chain length on overall stability is subtle, with a general
trend favoring longer chains that is less pronounced than it
was in our earlier guanidine systems. In some contexts,
however, chain length can be significant. Both the Ur/Cit
and Ur*/Cit complexes are much more stable (∆Tm ) 8 °C
in each case) when the shorter chain is attached to the acidic
parent sequence (i.e., complexes formed using pUrE or pUr*E
rather than pUrK or pUr*K). The analogous experiments with
buried guanidine pairs revealed a much lower preference for
one configuration over the other.5
After establishing the fundamental viability of urea-
substituted core residues, we began to explore interactions
between each of the three urea side chains and other
known polar groups. To begin, we paired each urea with
one of the three guanidinylated residues from our earlier
work (positioning the guanidine 1-3 CH2 groups from
the backbone).5 Thermal unfolding CD curves for the 18
possible combinations (nine each with the urea residues
on the acidic or basic peptides, respectively) exhibit
cooperative transitions and a broad range of melting
temperatures (Tm ) 51-75 °C).10
These guanidine/urea pairs exhibit an interesting preference
for parent sequence identity, with universally higher Tm
values observed when the guanidine residue is on the basic
rather than acidic peptide. The strength of this effect is quite
variable, with ∆Tm ranging from 2 to 13 °C.10 A similar
preference is exhibited by Gdn/Asn pairs but with less
variability (∆Tm ) 6-10 °C).5 The source of this selectivity
is not obvious but may be useful as an empirical guide to
future designs.
Figure 3. Urea/polar group interactions. Thermal stability of
heterodimers formed between the indicated basic urea peptides and
acidic peptides bearing a variety of polar groups.6
affinity partners, but similar length Asp and Glu residues
were only a bit better. The neutral Asn contact consistently
gives rise to the most stable complexes, but overall discrimi-
nation between groups tends to compress as the urea side
chain lengthens, as evidenced by the similar affinity of pCitK
for either pArgE or pAsnE. Even pGdE and pGd*E are
reasonable partners for pCitK, emphasizing that subtle
structural changes can materially alter recognition profiles.
With all of these new buried polar group interactions
catalogued, we turned our attention to their implementation
as directing elements in multicomponent mixtures. The
preference for self-recognition in the urea-functionalized
peptides suggested that a urea/urea pair might be added to
already successful four-component systems. The resulting
six-peptide mixture can form nine different complexes that
differ only by buried core residue juxtaposition (pairing each
acidic and basic peptide). We hoped that orthogonal recogni-
tion would winnow observed dimers down to the designed
three.
Given the selectivity in Gdn/Asn pairs, we next investi-
gated heterodimers positioning each urea derivative opposite
an asparagine. In contrast to the interaction of either group
with guanidines, amide/urea pairs were insensitive to the
parent strand, producing stable complexes in each case
(Tm ) 67-71 °C).10
Finally, we evaluated mixtures of each basic urea peptide
and either pAspE or pGluE, containing the indicated car-
boxylic acid side chain at position 14. Again, stable structures
were observed (Tm ) 55-67 °C), albeit somewhat less stable
To determine binding preferences, we employed an affinity
assay in which one peptide of the complex to be tested is
derivatized with an N-terminal GlyGly(His)6 sequence. An
equimolar mixture of the tagged peptide and its intended
Org. Lett., Vol. 10, No. 17, 2008
3799

binding partner(s) is then exposed to His-binding Ni-NTA
agarose beads. Following supernatant removal and washing,
bound material is recovered by treatment with imidazole
buffer, and each fraction is analyzed by HPLC.10 Untagged
peptide(s) stick to the beads (and appear in the elution
fraction) only by virtue of a specific interaction with the
tagged one. Those with no such affinity remain in the original
supernatant.
Impressively, appropriate six-component systems do in-
deed form three independent heterodimeric coiled-coils, with
little or no contamination from competing complexes (Figure
4). Mixtures of pAsnK/pAsnE, pCitK/pCitE, and pGdnK/pAspE
Figure 5. Unsuccessful six-component recognition experiment. An
equimolar mixture of pAsnK/pAsnE/pCitK/pCitE/pArgK/pGluEHis fails
to exhibit specific recognition. The elution fraction contains
substantial amounts of all three basic peptides, demonstrating that
Glu is not an appropriate binding partner for Arg in this context,
despite the success of Asp. Initial solutions were prepared as in
Figure 4.
Despite the subtle structural change, essentially all specificity
is removed. Each of the two other possible binding partners
for each component of the tagged dimer are observed in both
supernatant and elution fraction.
The results above dramatically expand the capacity of
simultaneous recognition designs in dimeric coiled coil
formation. Buried ureas are compatible with exceptionally
stable heterodimers, and the peptides are trivially synthesized.
Ureas are also good partners for guanidines, amides, and
acids, enabling many new designs. Although dimer stability
generally increases with polar side chain length, the cor-
relation is loose, and considerable Tm variance is observed
with subtle modifications. This permits need-specific tuning
of complex stability. As a demonstration that new polar pairs
enable otherwise challenging recognition events, we have
demonstrated simultaneous and specific recognition of up
to three distinct heterodimers driven solely by core matchups.
Given the ubiquitous application of coiled coil formation in
fundamental and applied contexts, this capacity should find
considerable use.
Figure 4. Ni-NTA analysis of six-component self-assembly. HPLC
analysis of an equimolar pAsnKHis/pAsnE/pCitK/pCitE/pArgK/pAspE
mixture demonstrates specific recognition: the elution fraction is
dominated by Asn peptides. The initial mixture was 20 µM total
peptide, pH 7.0, 150 mM NaCl, 10 mM phosphate. See Supporting
Information for the other successful cases.
were investigated (pGdnK ) any of the three guanidine
peptides). The four noncitrulline components were shown
previously to be specific, and in this case again tagging
pAsnK peptides affords an elution fraction containing es-
sentially only pAsnE. All four of the other peptides remain
in the supernatant. Separate analysis demonstrates that each
undesired dimer is intrinsically viable absent the other
peptides and that the four supernatant components exhibit
high fidelity as well.10 The overall system thus displays
impressive selectivity. The specific guanidine peptide seems
relatively unimportant.11
Acknowledgment. This work was supported by an NSF
CAREER award to A.J.K. (CHE-0239275).
The difficulty in constructing a viable six-component
recognition system is underscored by replacing the pAspE
peptide in the successful mixture with pGluE (Figure 5).
Supporting Information Available: Detailed experimen-
tal procedures, descriptions of urea chemistry, additional CD,
Ni-NTA, and analytical ultracentrifugation data. This material
is available free of charge via the Internet at http://pubs.acs.org.
(11) The results seem a bit cleaner with pArgK, though the four-
component analysis of the non-asparagine peptides is slightly less selective
in that case. See Supporting Information for details.
OL801461A
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Org. Lett., Vol. 10, No. 17, 2008