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 DNA

E

Q

upon recognition of their cognate bindingB

L

A

R

H

sequence (TGACTCA). C-terminal of this

M

K

Y

T

basic region is the leucine zipper (coiled coil)

V

I

D

region that is responsible for mediating

N

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 using

PYMOL

(DeLano Scientific; http://

d

cJun

a ’

pymol.sourceforge.net/). (B) A helical wheelcFos

A D T K

JunW

f ’

FosW

E K

representation highlighting the interaction

E S A E

JunW

CANDI

E K

f

FosW

C

patterns for the various heterodimers. Resi-cJun(R) FosW

Core

J (R)

a

FosW(E)dues for cJun (left) and cFos (right) are col-

d ’

oured black. Residues for JunW, JunW

CANDI

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,FosW

Core

and FosW(E) that differ from

Q

I

I

L

D

R

D

those of cFos are shown as blue, green and

A

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 GAPJunW

CANDI

AS AAELEER AKTLKAE IYELRSK ANMLREH IAQL GAPFosW

Core

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 m

M

Tris, 200 m

M

NaCl, 1 m

M

EDTA and 5 m

M

b-mercap-toethanol at pH 8) varied from this study.

this analysis, however, we have focused entirely on the

the overall free energy of binding for the wild-type leu-

unmodified coiled coil region of the wild-type AP-1

cine zipper complex is driven by a strong entropic com-

ponent. Moreover, as is the case for the parent AP-1

protein. This coiled coil dimerization motif is 4.5 hept-

leucine zipper, our antagonists are predicted to form a

ads in length. We find that the free energy of binding

helical structure that gives rise to a coiled coil with

is driven predominantly by a favourable entropy (TDS;