P05412) ANDC-FOS(UNIPROTKB
8077940:c-Jun(uniprotkb:P05412) andc-Fos(uniprotkb:P01100)bind(MI:0407) bycirculardichroism(MI:0016)
Introduction
The transcriptional regulator activator protein-1 (AP-1)
form different heterodimers, which in turn have differ-
ent expression patterns depending on the tissue. AP-1
generally consists of heterodimers of the Jun (e.g. cJun,
is responsible for the regulation of a number of key
JunB, JunD) and Fos (e.g. cFos, FosB, Fra1, Fra2)
families of proteins. Different homologues combine to
genes that include cyclin D1 and interleukin-2, and is
AbbreviationsAP-1, activator protein-1; CANDI, competitive and negative design initiative; ITC, isothermal titration calorimetry; PCA, protein-fragmentcomplementation assay; PPI, protein–protein interaction.AFig. 1. (A) The structure of the native DNA-bound cJun–cFos AP-1 bZIP domain (PDBcoordinates 1FOS) [7] containing the bZIPregion of the two proteins. cJun is shown inred and cFos in blue. The ‘basic’ N-terminalregions are rich in arginine and lysine andare responsible for scissor gripping the DNAE
Q
upon recognition of their cognate bindingBL
A
R
H
sequence (TGACTCA). C-terminal of thisM
K
Y
T
basic region is the leucine zipper (coiled coil)V
I
D
region that is responsible for mediatingN
Q
D
R
K
E
R
N
R
L
dimerization of the two chains, and is there-e ’
b ’
g
fore the focus of this study. The figure cre-c
ated usingPYMOL
(DeLano Scientific; http://d
cJuna ’
pymol.sourceforge.net/). (B) A helical wheelcFosA D T K
JunWf ’
FosWE K
representation highlighting the interactionE S A E
JunWCANDI
E K
f
FosWC
patterns for the various heterodimers. Resi-cJun(R) FosWCore
J (R)a
FosW(E)dues for cJun (left) and cFos (right) are col-d ’
oured black. Residues for JunW, JunWCANDI
c ’
b
e
A
V
b
I
and cJun(R) that differ from those of cJun’
g’
are shown as blue, green and red,respectively. Similarly, residues for FosW,FosWCore
and FosW(E) that differ fromQ
I
I
L
D
R
D
those of cFos are shown as blue, green andA
Q
red, respectively.connected to a number of cell signalling cascades. It
DNA binding and a coiled coil (leucine zipper) region
has consequently been demonstrated that AP-1 upreg-
that is known to mediate dimerization of the two
ulation is involved in a number of diseases, including
chains. Developing rules that can assist in the discov-
ery of new binding partners for coiled-coil-containing
cancer [1–3] bone disease (e.g. osteoporosis) and
proteins therefore has great potential for influencing
inflammatory diseases such as rheumatoid arthritis and
biology by elucidating stable and specific protein–pro-
psoriasis [4–6]. Thus, peptides capable of specifically
tein interactions (PPIs) [8]. We have consequently
sequestering key components of AP-1, and that there-
derived several peptides, based upon the coiled coil
fore prevent its function, show great promise as the
regions of AP-1, that are able to bind to the corre-
starting point for drugs to combat a number of dis-
sponding coiled coil regions of key AP-1 homologues
eases. The native AP-1 dimer (Fig. 1) consists of a
and prevent them from binding to DNA via their basic
transactivation domain, a basic domain, rich in lysine
region. Thus, these antagonists have the potential to
and arginine residues, that is responsible for mediating
sequester these proteins as nonfunctional heterodimers
Thermodynamics of binding
to prevent binding to native partners. The first of these
peptides was generated by semirational design using
To enable us to address the question of a common
the native binding partner as a scaffold. Degenerate
underlying mechanism by which all of these antago-
codons important in dimerization were introduced and
nists achieve high interaction affinity, we decided to
a protein-fragment complementation assay (PCA)
use CD data and isothermal titration calorimetry
[9,10] was undertaken to screen the resultant library
(ITC) to split the free energy of binding into its com-
and single out peptide sequences capable of generating
ponent parts, the enthalpy (DH) and the temperature
multiplied by the entropic contribution (TDS) accord-
an interaction with the target protein. This ensured
ing to the relationship:
that only library members that bound to the target
generated colonies under selective conditions. Growth
DG
bind
¼ DH TDS ð1Þ
competitions then ensured that only those PPIs of
Where a negative DG
bind
value represents a sponta-
highest affinity were enriched. The peptides, JunW and
neous reaction that is favourable, DH represents the
FosW, bound to cFos and cJun, respectively, with
strength of the target–antagonist complex relative to
much higher interaction stability than the parent pro-
tein [11]. In order to increase the specificity of PCA-
those of the solvent and includes electrostatic bonds,
van der Waal’s interactions and hydrogen bond forma-
generated PPIs, we incorporated a competitive and
tion. A negative DH value is representative of a
negative design initiative (CANDI) into the screen.
favourable enthalpic contribution to the reaction. By
CANDI is used to ensure that the energy gap between
contrast, a positive TDS value represents a favourable
desired and nondesired complexes is maximized and
entropic contribution. Favourable entropy can come
works by including sequences competing for an inter-
action with either the target and ⁄ or the library member
from hydrophobic interactions that release water mole-
in the bacterial selection [12,13]. Library members that
cules upon their formation as well as minimal loss in
conformational freedom. Although binding affinity can
bind to the competitor, are promiscuous in their bind-
be optimized by either enthalpic or entropic improve-
ing selection or cannot compete with the competitor–
target complex are subsequently removed from the
ments, so long as they are not compensated for by
opposite entropic or enthalpic changes [16,17], optimi-
bacterial pool. Using the PCA–CANDI technique, we
zation of the binding energy via a negative enthalpic
generated a peptide, JunW
CANDI
, that is specific for
term is favoured. However, optimizing noncovalent
cFos even in the presence of a cJun competitor. This
bonds is extremely difficult to achieve by rational
is in sharp contrast to JunW, which binds with high
affinity to both cJun and cFos. This study offers the
design, because it is often accompanied by entropy
compensation. By studying a range of antagonists that
possibility to look at the underlying thermodynamic
have been designed or selected by enriching the highest
signature behind these two binding events. Libraries
affinity binding partners from libraries that target cJun
based on the cJun–FosW peptide have also been cre-
and cFos, it is anticipated that we can split the free
ated with both core and electrostatic semirandomiza-
energy of binding into its thermodynamic components
tions. Using competitive growth competitions, it was
found that the winner of the core randomization,
to investigate whether there is a thermodynamic profile
that is common to all of these molecules.
FosW
Core
, was able to bind to cJun specifically in the
presence of competing Fos homologues [14]. The
FosW
Core
library was based upon FosW and con-
Results
tained 12 residue options (codon NHT = F, L, I, V,
S, P, T, A, Y, H, N or D) at four of five a position
We used ITC to extract the thermodynamic parameters
that make up the overall free energy of binding (DG
bind
)
residues. This study reflected the fact that core resi-
dues impose large energetic changes, with consequent
for our antagonist–peptide complexes. The antagonists
(see Table 1 for sequences and Fig. 2 for example ITC
growth competitions, suggesting that they also have
the ability to impart specificity in instances where
profiles) have previously been shown to be capable of
sequestering cJun or cFos using a variety of techniques,
electrostatic options are insufficient. Finally an elec-
including CD thermal denaturation studies [11,12,20],
trostatically enhanced dimer, cJun(R)–FosW(E), has
kinetic folding studies [15,21] and native gel analysis
been previously studied to dissect the free energy of
[12,15]. We observe that the enthalpic component is
binding into its component steps, and was found to
strongly favoured for our antagonist–target complexes
have achieved increased equilibrium stability as a
and that the change in entropy is unfavourable. How-
result of large decrease in the dissociation rate of the
complex [15].
ever, in contrast to Seldeen et al. [18], we observe that
Table 1. Peptide sequences and the sequences used by Seldeenet al.[18], which lack N and C capping motifs and contain an 11.7 kDa thi-oredoxin motif fused to the N-terminus and a hexahistidine tag at the C-terminus, separtated by thrombin cleavage sites.SequenceNameabcdefg abcdefg abcdefg abcdefg abcdcJun AS IARLEEK VKTLKAQ NYELAST ANMLREQ VAQL GAPcFos AS TDTLQAE TDQLEDE KYALQTE IANLLKE KEKL GAPFosW AS LDELQAE IEQLEER NYALRKE IEDLQKQ LEKL GAPJunW AS AAELEER VKTLKAE IYELQSE ANMLREQ IAQL GAPJunWCANDI
AS AAELEER AKTLKAE IYELRSK ANMLREH IAQL GAPFosWCore
AS IDELQAE VEQLEER NYALRKE VEDLQKQ AEKL GAPcJun(R) AS IARLRER VKTLRAR NYELRSR ANMLRER VAQL GAPFosW(E) AS LDELEAE IEQLEEE NYALEKE IEDLEKE LEKL GAPLZ (cJun)a
Trx-IARLEEK VKTLKAQ NSELAST ANMLREQ VAQLKQK-(His)6
LZ (cFos)a
Trx-TDTLQAE TDQLEDE KSALQTE IANLLKE KEKLEFI-(His)6
a
Seldeenet al.[18,19] generated 28mers with peptides fused to an 11.7 kDa N-terminal thioredoxin (Trx) tag to assist with solubility andexpression, as well as a C-terminal (His)6
-tag. Both tags were additionally separated by thrombin sites (LVPRGS) which upon cleavagecaused significant destabilization of the peptides. Their experimental conditions (50 mM
Tris, 200 mM
NaCl, 1 mM
EDTA and 5 mM
b-mercap-toethanol at pH 8) varied from this study.