Báo cáo Y học: Interactions between the Fyn SH3-domain and adaptor protein Cbp/PAG derived ligands, effects on kinase activity and affinity docx

Interactions between the Fyn SH3-domain and adaptor
protein Cbp/PAG derived ligands, effects on kinase
activity and affinity
Silje A. Solheim
1,2
, Evangelia Petsalaki
3
, Anne J. Stokka
1,2
, Robert B. Russell
3
, Kjetil Taske
´
n
1,2
and Torunn Berge
1,2
1 The Biotechnology Centre of Oslo, Norway
2 Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo, Norway
3 European Molecular Biology Laboratory, Heidelberg, Germany
Tyrosine phosphorylation is one of the key regulatory
protein modifications in multicellular organisms and
tightly controls and coordinates a wide range of cellu-
lar responses such as growth, metabolism, tissue
repair, migration and apoptosis [1–3]. Phosphorylation
modulates enzymatic activity as well as creating new
binding sites for the recruitment of active molecules
into signalling complexes, and assists in building
dynamic networks for the transduction of information
from the extracellular environment to intracellular
signalling pathways. Accurate and specific processing

of information is vital to maintaining cellular homeo-
stasis, and errors in signal transduction pathways are
linked to a range of diseases such as cancer, auto-
immunity and diabetes [4,5].
Tyrosine phosphorylation is a reversible modifica-
tion, regulated by protein tyrosine kinases (PTKs) and
phosphatases (PTPs) [6,7]. PTKs of the Src kinase
family employ a well-conserved modular arrangement
of interaction domains in the regulation of kinase
Keywords
kinase activity; proline-rich motifs; protein–
protein interactions; SH3 domain; tyrosine
phosphorylation
Correspondence
T. Berge, The Biotechnology Centre of Oslo,
Gaustadalleen 21, N-0319 Oslo, Norway
Fax: +47 2284 0501
Tel: +47 2284 0519
E-mail:
Website:
(Received 8 May 2008, revised 24 June
2008, accepted 4 August 2008)
doi:10.1111/j.1742-4658.2008.06626.x
Csk-binding protein ⁄ phosphoprotein associated with glycosphingolipid-
enriched domains is a transmembrane adaptor protein primarily involved
in negative regulation of T-cell activation by recruitment of C-terminal Src
kinase (Csk), a protein tyrosine kinase which represses Src kinase activity
through C-terminal phosphorylation. Recruitment of Csk occurs via SH2-
domain binding to PAG pTyr317, thus, the interaction is highly dependent
on phosphorylation performed by the Src family kinase Fyn, which docks

onto PAG using a dual-domain binding mode involving both SH3- and
SH2-domains of Fyn. In this study, we investigated Fyn SH3-domain bind-
ing to 14-mer peptide ligands derived from Cbp ⁄ PAG-enriched micro-
domains sequence using biochemical, biophysical and computational
techniques. Interaction kinetics and dissociation constants for the various
ligands were determined by SPR. The local structural impact of ligand
association has been evaluated using CD, and molecular modelling has
been employed to investigate details of the interactions. We show that data
from these investigations correlate with functional effects of ligand binding,
assessed experimentally by kinase assays using full-length PAG proteins as
substrates. The presented data demonstrate a potential method for modula-
tion of Src family kinase tyrosine phosphorylation through minor changes
of the substrate SH3-interacting motif.
Abbreviations
Cbp ⁄ PAG, Csk binding protein ⁄ phosphoprotein associated with glycosphingolipids-enriched microdomains; Csk, C-terminal Src kinase; GST,
glutathione S-transferase; PPII, polyproline type II; PRD, proline-rich domain; PTK, protein tyrosine kinase; SFK, Src family kinase; SH2, Src
homology 2; SH3, Src homology 3; TCR, T-cell receptor.
FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS 4863
activity and inhibition, as well as in the combinatorial
assembly of active signalling complexes. The common
structure consists of an N-terminal membrane-target-
ing region (SH4), a Src-homology 3 (SH3) domain and
a Src-homology 2 (SH2) domain, capable of binding
to proline-rich motifs and phosphotyrosine residues,
respectively. These interaction domains are followed
successively by a tyrosine kinase (SH1) domain [8,9].
In addition, Src family kinases (SFKs) contain both a
C-terminal auto-inhibitory phosphorylation site and an
activating auto-phosphorylation site in the kinase
domain. Interactions occur frequently via individual

domains; however, both domains may also cooperate
to facilitate specific and stable complex formation [10–
12].
In T cells, the SFKs Lck and Fyn have central roles
in early signal transduction events as the most proxi-
mal signalling molecules to be activated downstream
of the T-cell receptor following direct interaction
between the receptor and peptide–MHC complexes on
antigen presenting cells. This activation leads to a cas-
cade of tyrosine phosphorylation-dependent signalling
pathways [2,3,13]. Fyn associates with and phosphory-
lates the transmembrane adaptor protein Csk-binding
protein ⁄ phosphoprotein associated with glycosphingo-
lipid-enriched microdomains (Cbp ⁄ PAG) [14], which is
localized to lipid rafts by palmitoylation anchoring
[15]. The adaptor functions primarily as a negative reg-
ulator of SFKs and T-cell activation via recruitment of
C-terminal Src kinase (Csk) to PAG pTyr317 (human)
[16–18], however, the regulatory role of PAG may be
more complex as the adaptor may also act as an acti-
vating partner of Lyn, an SFK, and pSTAT3 in B-cell
lymphomas, as suggested by Tauzin et al. [19].
The involvement of either SH3 or SH2 domains has
been discussed for the association of Fyn with PAG
[20,21]; in fact, Fyn utilizes both domains in this inter-
action, as we have demonstrated previously [22]. Bind-
ing of the Fyn SH3-domain to the first proline-rich
region of PAG is essential for the initiation of PAG
tyrosine phosphorylation, which occurs via a proces-
sive phosphorylation mechanism [23]. This sub-

sequently allows binding of the Fyn SH2-domain to
PAG Tyr163 or pTyr181 and results in a dual-domain
docking, enhancing the affinity of the Fyn–PAG inter-
action and rendering Fyn insensitive to negative regu-
lation by Csk (Fig. 1A).
In this study, we explore further the initial step of
the Fyn–PAG association; binding of the kinase SH3-
domain to the first proline-rich region of PAG. Full-
length PAG variants, developed using a 2D peptide
array approach, have previously been employed to
demonstrate the functional effects of the Fyn–PAG
complex in T cells [22]. Using full-length proteins as
templates, we created 14-mer peptide ligands (Fig. 1B),
one corresponding to the wild-type PAG interaction
region (PRD1), and two structural variants of this
sequence with low (PRD1-RLP*) and high (PRD1-
super) affinity for the Fyn SH3-domain, respectively.
PAG also has a second proline-rich region, known to
interact with the SH3-domain of the related kinase
Lyn [24], and a 14-mer peptide (PRD2) containing this
binding site was included as a control. We conducted
binding assays of peptide ligands using the isolated
Fyn SH3-domain (Fig. 1C) as well as full-length Fyn
affinity precipitated from human primary T-cell
lysates. Kinetics and specificity of Fyn SH3-domain
interactions were determined using SPR, and CD spec-
troscopy was used to assess local structural changes
upon SH3-domain binding. The tertiary structure of
the Fyn SH3-domain complexed to ligand is known
both from X-ray diffraction and NMR studies [25,26],

A
C
B
Fig. 1. The Fyn-PAG interaction. (A) Schematic representation showing Fyn binding to the transmembrane adaptor PAG via SH3- and
SH2-domain interactions. (B) The 14-mer peptide ligands used in this study include the first (PRD1) and second (PRD2) proline-rich regions
of PAG as well as variants of the first region (PRD1-RLP* and PRD1-super) developed using 2D peptide arrays [22]. (C) Secondary structure
elements of the Fyn SH3-domain. The five b strands, numbered consecutively, are separated by the interaction loops (bold) making primary
contacts with the ligand. Trp119 (*) is a highly conserved residue among SFK SH3-domains.
Modulation of the Fyn SH3–PAG interaction S. A. Solheim et al.
4864 FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS
and this information was used to model the pep-
tide ⁄ SH3-domain complexes in silico to investigate
details of the ligand interactions. We show that
changes in affinity and secondary structure have signif-
icance for the functional effects of ligand binding,
assessed experimentally by kinase assays using full-
length PAG proteins as substrates.
Results and Discussion
Phosphorylation of the adaptor protein PAG is depen-
dent on an initial interaction between the first proline-
rich region of PAG and the SH3-domain of the kinase
Fyn. Our earlier investigations have highlighted the
importance of the Fyn SH3-domain interaction in initi-
ating the association with PAG, and how this affects
tyrosine phosphorylation and thereby the functionality
of PAG as a negative regulator of T-cell activation
through recruitment of Csk, dependent on PAG
pTyr317 [22]. In this study, we recreated this associa-
tion in vitro using isolated Fyn SH3-domains and syn-
thetic peptide ligands containing wild-type PAG

sequences as well as structural variants developed
using a 2D peptide array approach, a rapid and semi-
quantitative approach for evaluation of amino acid
substitutions of all residues in a defined region.
The Fyn–PAG interaction: association via the Fyn
SH3-domain
Several studies have focused on the identification of
high-affinity ligands for SH3-domains, either through
phage display or combinatorial peptide libraries [27–
29]. Combined with data from NMR and X-ray crys-
tallography [25,26,30,31], these reports have revealed
in detail many of the requirements of SH3-domain
ligand binding, and optimal core recognition motifs
have been established for several kinase SH3-domains,
such as RPLPPLP for Src, Fyn, Lyn and phosphati-
dylinositol 3-kinase. This motif is a class I SH3
consensus motifs (RxxPxxP), characterized by an
N-terminal arginine residue known to form an orienta-
tion-determining salt bridge with a key aspartate resi-
due [27,28,32]. Interestingly, the Fyn SH3 domain
contains two such adjacent residues Asp99 and
Asp100, which may both participate in formation of
this salt bridge [33]. Using 2D peptide array analyses,
the minimal interaction sequence of the wild-type
PAG-derived peptide PRD1 has been established to
RELPRIP [22], i.e. four residues in common with the
phage display motif, whereas the high-affinity PRD1-
super peptide (Glu132Pro, Pro135Arg of PAG, human)
of this study matched the minimal motif with five
common residues. By contrast, the low-affinity

sequence PRD1-RLP*, lacked critical amino acids by
three alanine substitutions of the minimal motif
(Arg131, Leu133 and Pro134 of PAG, human), and
PRD2 (PPPVPVK), the peptide corresponding to the
second proline-rich region of PAG, had only two
residues in common with the minimal core motif.
To analyse further the implications of PAG binding
to the Fyn SH3-domain, 14-mer peptides were synthes-
ised consisting of these sequence variants of the inter-
action site (PRD1, PRD1-RLP* and PRD1-super)as
well as PRD2 (Fig. 1B). Interaction data obtained
using full-length proteins were first verified by per-
forming biotin–streptavidin affinity precipitations on
human T-cell lysates using biotin-tagged peptide
ligands (Fig. 2). The pull-down assays demonstrated a
significant reduction in binding using the PRD1-RLP*
peptide, whereas the PRD1-super peptide, developed as
a high-affinity Fyn SH3 binder using the 2D peptide
array approach, displayed an  2.5-fold increase in
Fyn binding relative to the wild-type (PRD1) peptide.
Interaction with the first proline-rich sequence was
exclusive as no association was observed in pull-down
assays performed with the peptide PRD2 containing
the second PAG proline-rich domain.
Conformation of peptide ligands in solution
Polyproline sequences are known to adopt a distinct
secondary structure in aqueous solutions due to the
unique conformational properties of the cyclic imino
nature of proline residue [34,35]. This structure, a
polyproline type II helix (PPII), has a specific geo-

metry exploited in SH3-domain ligand binding that
enables direct interactions with the hydrophobic bind-
ing grooves of this domain. Using CD spectroscopy,
we investigated the extent of PPII helix conformation
of the peptide ligands PRD1, PRD1-RLP*,
Fig. 2. Fyn interacts with the first proline rich region in PAG.
Lysates from human primary T cells were incubated with the indi-
cated biotin-linked peptides and complexes precipitated using
excess streptavidin beads. Interactions were evaluated by western
blot analysis using anti-Fyn IgG.
S. A. Solheim et al. Modulation of the Fyn SH3–PAG interaction
FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS 4865
PRD1-super and PRD2 (Fig. 3). In water, typical CD
spectra of the PPII helix show a negative band at 200–
205 nm and a positive band at 217–225 nm [36].
Although the positive band was absent from our sam-
ples, all four peptides displayed strong negative bands
at  201 nm, indicating that they are not completely
unstructured in aqueous buffer. The negative maxima,
believed to arise from the extent of PPII conformation,
was found to correspond with the number of proline
residues in each peptide sequence (PRD2 >
PRD1 = PRD1-super > PRD1-RLP*). The absence
of a characteristic positive shoulder from all spectra
was thought to arise from the presence of other sec-
ondary structure elements in the samples [34,35].
Effects of PAG–Fyn SH3 interaction modulation
The functional effects of modulating the affinity of
the PAG–Fyn SH3 interaction were examined in an
in vitro kinase assay using full-length, recombinant

PAG protein and active Fyn kinase. In T cells, PAG
has a dual role as both a ligand and a substrate for
Fyn, and with nine potential tyrosine phosphorylation
sites the adaptor is an excellent subject for quantitative
analysis of phosphorylation effects mediated via SH3-
domain interactions.
In a pulse-chase assay, active Fyn and recombinant
PAG were incubated on ice with a low concentration
of radioactive ATP. After 10 min, a large excess of
ATP was added and the phosphorylation process was
continued with aliquots withdrawn at the indicated
time points as illustrated for wild-type PAG in
Fig. 4A. Phosphorylated PAG species appeared as dis-
tinct bands, with the slower migrating species corre-
sponding to more heavily phosphorylated protein
accumulating over time. Quantitative analyses of PAG
phosphorylation of the three variants were performed
over a time course of 5 min by measuring pixel inten-
sity of the highest band at each time point relative to
fully phosphorylated wild-type PAG (Fig. 4B). The
wild-type PAG protein was rapidly phosphorylated by
Fyn, reaching the highest molecular mass species after
 1 min. The full-length high affinity SH3-binder,
superPAG, was phosphorylated in a similar fashion;
however, the accumulation of protein tyrosine phos-
phorylation appeared more pronounced (approxi-
mately twofold). The rapid phosphorylation progress
and the appearance of discrete bands, as illustrated in
Fig. 4A, are typical of a processive phosphorylation
process [22,23,37], governed by an initial interaction

between the Fyn SH3-domain and PAG. This binding
mode is indispensable for rapid and efficient PAG
phosphorylation, as full-length PAG-RLP* with a
PRD1
PRD1-RLP*
PRD1-super
PRD2
Wavelength (nm)
200 210 220
230
240 250 260
[θ] (deg·cm
2
·dmol
–1
) (x10
5
)
50
0
–50
–100
–150
–200
–250
–300
Fig. 3. Far-UV CD spectroscopy of the PAG-derived peptide
ligands. Spectra were recorded in 10 m
M Hepes (pH 7.4), 150 mM
NaCl at a sample concentration of 90 lM.

Time (min)
Relative intensity (%)
wt PAG
PAG RLP*
super PAG
1 2
3
4
5 0
0
50
100
150
200
250
300
75 kDa
0
20 40
60 80
120
Time
100
100 kDa
10 20
min
s
pPAG
A
B

PA G
Fig. 4. Kinetics of PAG phosphorylation by wild-type Fyn. In vitro
pulse-chase assays were performed using active Fyn kinase and
various full-length PAG constructs incubated on ice in the presence
of 80 n
M [
32
P]ATP[cP] for 10 min. Addition of 0.5 mM ATP initiated
the chase reaction, aliquots were withdrawn at specified time
points and reactions stopped by mixing with SDS ⁄ PAGE sample
buffer and boiling. Time points were resolved on SDS ⁄ PAGE gels
and detected using autoradiography. (A) Phosphorylation of wild-
type PAG using active Fyn kinase illustrating the autoradiography
gels used for quantification of phosphorylation levels. Coomassie
stain shows equal loading of PAG in each sample. (B) Tyrosine
phosphorylation effects of the PAG–Fyn SH3 interaction modula-
tion. Phosphorylation levels were quantified as pixel intensity of the
highest molecular species relative to phosphorylated wild-type (wt)
PAG at 5 min. Error bars show SEM over three repeat experiments.
Modulation of the Fyn SH3–PAG interaction S. A. Solheim et al.
4866 FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS
greatly reduced Fyn SH3-domain affinity showed a
significantly delayed tyrosine phosphorylation. Phos-
phorylation of the PAG-RLP* protein most likely fol-
lows a distributive course caused by random collisions
between the substrate and kinase. Efficient phosphory-
lation of PAG-RLP* consequently occurs only after
phosphorylation of Tyr168 and Tyr181, which accord-
ing to our interaction model [22] allows Fyn to dock
onto its substrate via SH2-domain binding, thus

switching to tyrosine phosphorylation in a processive
manner. Again, the interaction was deemed specific for
the first proline-rich PAG sequence, as all substrate
molecules included in the kinase assays contained an
unaltered second proline-rich region (amino acids 252–
265 of PAG, human) which did not appear to affect
tyrosine phosphorylation by Fyn in vitro.
Structural impact of Fyn SH3-domain ligand
binding
Far-UV CD was employed to characterize local struc-
tural changes in the Fyn SH3-domain following binding
of PAG-derived peptide ligands. CD spectra of the Fyn
SH3-domain alone (Fig. 5A) showed a minimum at
A
B
C
Fig. 5. Far-UV CD spectroscopy of the Fyn
SH3-domain interaction with PAG derived
ligands. (A) Far-UV CD spectra of the Fyn
SH3-domain, the PRD1 peptide and the SH3
domain-peptide complex measured in
10 m
M Hepes (pH 7.4), 150 mM NaCl. (B)
Spectra of SH3-domain ligand mixtures at a
ligand concentration range of 10–90 l
M.
Spectra of free peptide were subtracted
from those of the mixture. (C) Difference
spectra of the SH3-ligand mixtures [data
presented in B with spectra of the free

SH3-domain subtracted (a)b)].
S. A. Solheim et al. Modulation of the Fyn SH3–PAG interaction
FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS 4867
207 nm and two maxima at 221 and 236 nm, compara-
ble with previous reports on SFK SH3-domains [38,39].
Addition of peptide containing the wild-type binding
site (PRD1) resulted in a positive shift of the maxima
and an increase in positive ellipticity over the 220–
240 nm region, whereas the minimum was slightly blue-
shifted with an increase in negative ellipticity. The latter
observation may indicate an increase in PPII conforma-
tion caused by stabilization of the proline-containing
peptide upon binding to the SH3-domain [34].
Ligand binding over a concentration range of 10–
90 lm showed that the wild-type sequence and the
high-affinity SH3-binder, PRD1-super, induced a local
concentration-dependent structural change over the
same region (220–240 nm, Fig. 5B). Spectra for both
peptides were found to approach a saturation maxi-
mum. Increasing concentrations of the low-affinity
SH3-binder, PRD1-RLP*, did not affect secondary
structure, as estimated by CD measurements; neither
did the control peptide PRD2 containing the second
proline-rich region. The largest difference, Dh, in the
215–250 nm region was plotted for the highest peptide
concentrations (90 lm, Fig. 5C). This clearly revealed
that the relative local structural change was signifi-
cantly greater for the high-affinity binder, showing a
broad positive band with an ellipticity maximum cen-
tred at 225 nm. Positive ellipticity in this region has

been attributed to interactions with clustered aromatic
amino acids [39,40], which may involve tyrosine resi-
dues located in the interaction loops of the SH3-
domain. Other parts of the differential spectrum were
not significantly influenced by ligand binding to the
SH3-domain for either peptide (data not shown).
Determination of affinity and kinetic constants
Binding affinities and dissociation constants for ligands
derived from the first proline-rich domain of PAG
were analysed using SPR. To this end, we immobilized
Fyn SH3-fusion proteins to surfaces of biosensor
chips, as described above, and assayed for binding by
passing the peptides PRD1, PRD1-RLP* and PRD1-
super over immobilized proteins. Thus, a single chip
was used for all peptides, minimizing effects of varia-
tions in ligand concentration. Binding profiles
(Fig. 6A) obtained this way corresponded well with
AB
CD
Fig. 6. SPR measurements of the interaction between immobilized Fyn SH3-domains and peptides PRD1, PRD1-RLP* and PRD1-super. (A)
All three peptides were injected over the sensor chip at 100 l
M to reveal differences in binding. (B–D) Peptides (B, PRD1; C, PRD1-RLP*;D,
PRD1-super) were injected over the sensor chip for kinetic analysis of the SH3-domain interaction. Ligand binding curves for peptide concen-
trations of 3.1, 12.5, 50 and 200 l
M are shown. Binding was measured in resonance units (RU), where 1 RU corresponds to the binding of
1pgÆmm
)2
.
Modulation of the Fyn SH3–PAG interaction S. A. Solheim et al.
4868 FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS

results from pull-down assays using biotinylated pep-
tides. Approximately twice as much PRD1-super pep-
tide as the PRD1 peptide was found to bind to
immobilized SH3-domains over multiple experiments,
while virtually no binding to the immobilised proteins
was observed for the PRD1-RLP* peptide.
To determine association and dissociation rate con-
stants for the peptides, concentration series were
injected over the sensor chips (Fig. 6B–D, representa-
tive curves from a single run are shown). Kinetic data
from the binding curves were evaluated using a two-
state conformational change model, which was found
to provide a better fit than the 1 : 1 Langmuir model
based on analyses of residuals and v
2
values. Our
choice of model is backed by findings using CD spec-
troscopy (Fig. 5), which indicated that ligand binding
induced a structural change in the Fyn SH3-domain.
A summary of kinetic constants is given in Table 1.
Dissociation constants showed that the PRD1-super
peptide bound to the Fyn SH3-domain with approxi-
mately fourfold higher affinity than the wild-type inter-
action sequence. K
D
estimated for the wild-type PRD1
peptide was in the low micromolar range ( 4–7 lm)
in repeat experiments, of the same order of magnitude
as previous studies on SFK SH3-domains using similar
technology [28,39,41–43].

Molecular modelling of the Fyn SH3-domain and
PAG-derived peptides
The interaction pockets of the SH3-domain are made
by loops linking the individual b strands together (the
RT loop, the n-Src loop, and the 3
10
helix as indicated
in Fig. 1C), flanked by strands b4 and b5. The variable
loops n-Src and RT are principal determinants for
ligand recognition, orientation, and specificity of this
domain, with residues Tyr91 and Tyr137 forming
interaction pocket 1; Tyr93, Tyr137 and Trp119 form-
ing pocket 2, and the valley between n-Src and RT
loops included by Trp119 and Tyr132 making up the
third interaction pocket [31,44].
Molecular modelling of the ligand ⁄ SH3-domain
interactions were performed using the modeller soft-
ware as described above. Three available structures
(1fyn [25], 1azg [26] and 1a0n [26]) showing the Fyn
SH3 domain binding to peptides were superposed to
establish the positioning of the SH3 binding PxxP
motif and the alignment of our peptides to a molecular
model (Fig. 7). To model our peptides binding to the
SH3 domain, we selected the NMR structure 1azg of
the Fyn SH3 domain complexed to a proline-rich pep-
tide P2L (PPRPLPVAPGSSKT) corresponding to resi-
dues 91–104 of the p85 subunit [26]. P2L contains the
class I SH3 consensus motif RxxPxxP, sharing
sequence properties with both PRD1 and PRD1-super.
The first four residues of each peptide (CHQS) were

common to all models, thus, all peptide sequences were
numbered starting with the first residue of the model
sequence, arginine for PRD1 and PRD1-super and
alanine for PRD1-RLP*, respectively.
Alignment of the PRD1 peptide containing the wild-
type PAG interaction sequence binding to Fyn SH3
(Fig. 7B), predicted both hydrophobic and electrostatic
interactions involved in formation of the complex. The
model showed peptide residues Pro4 and Pro7 fitting
well into binding grooves 1 and 2 created by the n-Src
loop and the 3
10
helix, stabilized by stacking interac-
tions of the pyrrolidine ring of Pro7 with the aromatic
side chain of Tyr137. Modelling of the PRD1-super
peptide, which included two amino acid substitutions
(Glu2Pro and Pro8Arg), again showed main interac-
tions via the core residues for binding (Arg1, Pro4 and
Pro7). The proline substitution (Pro2) positioned
directly after the arginine residue occupied pocket 3
and was stacked against the aromatic residue Trp119.
Previous reports have suggested that pocket 3, binding
arginine via acidic side chains, could favourably
accommodate binding of a proline residue in this way
[45], and it is likely that the proline substitution pro-
vides a major contribution to the increased affinity for
the PRD1-super peptide. Advantages of the Arg8
introduction were less obvious as alignment of flanking
residues correlated with greater flexibility in position-
ing; however, further stabilization of the complex

could be provided by H-bond interactions via water
molecules and other amino acids not evaluated in this
model.
Impact of the various amino acid substitutions was
demonstrated by superposing the variant peptides
PRD1-RLP* (green) and PRD1-super (blue) onto the
wild-type PRD1 peptide (beige) (Fig. 7C). The mod-
elled structures showed that alanine substitution of
Table 1. Kinetic constants for PAG-derived ligands and Fyn
SH3-domain interactions
a
.
k
a1
(M
)1
Æs
)1
)
k
d1
(s
)1
) k
a2
(s
)1
)
k
d2

(s
)1
) K
D
(M)
PRD1 7.7 · 10
4
0.41 2.5 · 10
)4
0.01 5.2 · 10
)6
PRD1-super 6.8 · 10
4
0.1 6.9 · 10
)4
0.003 1.2 · 10
)6
a
Apparent association (k
a
) and dissociation (k
d
) rate constants and
affinity constants (K
D
) were calculated from three independent
experiments. Numbers in the table are shown for one representa-
tive experiment. No kinetic data could be obtained for the Fyn
SH3 ⁄ PRD1-RLP* peptide interaction. SD < 1% for all constants,
apart from PRD1 k

a2
and k
d2
where SD < 5%.
S. A. Solheim et al. Modulation of the Fyn SH3–PAG interaction
FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS 4869
Arg1 appeared to reduce interactions with acidic resi-
dues of pocket 3 (e.g. residues surrounding Trp119).
The substitution removes the arginine residue of the
RxxPxxP motif, thus abrogating the formation of the
salt bridge with Asp99, which defines the orientation
of the peptide ligand [33]. Alanine substitution of
Pro4 eliminated its stacking interactions with the cen-
tral tryptophan residue Trp119, believed to exten-
sively reduce the ability of the PRD1-RLP* ligand to
form a stable complex with the Fyn SH3-domain.
The overlay structures emphasised the significance of
the PRD1-super Pro2 substitution, revealing a good
fit adjacent to Arg1 where interactions with aromatic
and acidic residues such as Trp119, Asp99 and
Asp118 of the SH3-domain were likely. It is highly
probable that the predicted interactions outlined
above result in the local structural changes in the
Fyn SH3-domain as observed by CD measurements,
and that these changes affect binding affinity as eval-
uated by SPR.
Conclusions
We have described in detail the interactions of a nat-
ural Fyn SH3-domain ligand extracted from the adap-
tor protein PAG and low- and high-affinity variants of

this sequence developed using a 2D array approach.
Interaction kinetics and local structural impact of these
ligands binding to the Fyn SH3-domain were analysed
using SPR and CD, respectively, and the results of
these investigations related to functional effects of
ligand binding, assessed experimentally by kinase
assays using full-length PAG proteins containing the
modified binding motifs as substrates. In summary,
our data demonstrate that substrate phosphorylation
may be modified through minor changes in the
SH3-domain interacting motif.
Optimal binding sequence motifs have previously
been found using random or biased library
approaches, performed with several cycles of selection
for optimalization [27–29,46]. In this study, we used a
Fig. 7. Molecular models showing the Fyn
SH3-domain complexed to PAG-derived
peptides. (A) Superposition of the three
available PDB structures for the Fyn SH3
domain complexed to ligand peptides (1azg
(yellow) [26], 1fyn (green) [25] and 1a0n
(red) [26]). (B) Models of the Fyn SH3-
domain in complex with PRD1 and
PRD1-super peptides based on the NMR
structure 1azg [26]. The peptide is displayed
as sticks; the SH3-domain is shown as rib-
bons, while selected amino acids predicted
to be essential for interaction with the
ligand are shown as spheres. (C) Superposi-
tion of the PRD1-RLP* (green) and PRD1-

super (blue) peptides on the PRD1 (beige)
peptide complexed to the Fyn SH3-domain.
Modulation of the Fyn SH3–PAG interaction S. A. Solheim et al.
4870 FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS
natural ligand as a point of reference for the develop-
ment of high- and low-affinity ligands using a 2D pep-
tide array approach. This is a simple and rapid
method for analysis of individual amino acid substitu-
tions, and the high-affinity SH3-binder, PRD1-super,
developed using this strategy was found to have a dis-
sociation constant in the low micromolar range, com-
parable to that of the phage-display library peptide
VSL12 (1.2 versus 0.6 lm) [46].
In vivo manipulation of protein interaction domains
in this way could provide valuable insight into the
functional consequences of localized protein–protein
interactions. As an example, the high-affinity, full-
length PAG (superPAG) construct has been used in
T cells to reveal that the negative regulatory potential
of PAG is enhanced by this modification, consistent
with a higher degree of PAG phosphorylation and
concomitant Csk recruitment [22]. On a similar note, is
the kinase targeting strategy used by viruses such as
HIV and Herpesvirus saimiri which encode accessory
proteins containing proline-rich domains [47–49].
These proteins, Nef and Tip, respectively, interact with
kinase SH3-domains with high affinity to modify the
behaviour of virus-infected cells, and have been shown
to control both pathogenicity and T-cell proliferation.
This demonstrates the potential of manipulating signal-

ling pathways in vivo by creating high-affinity ligands
to compete with natural SH3-protein interactions.
Experimental procedures
Peptide synthesis
Peptides PRD1 (CHQSRELPRIPPES), PRD1-RLP* (CHQ-
SAEAARIPPES), PRD1-super (CHQSRPLPRIPRES) and
PRD2 (EEEAPPPVPVKLLD) were synthesized with or
without biotin-tags coupled to the N-terminus in-house or
purchased from AnaSpec Inc. (San Jose, CA, USA). Purity
was analysed by HPLC and mass spectroscopy. All pep-
tides were lyophilized prior to re-suspension in NaCl ⁄ P
i
,
and peptide concentration was determined using a Bio-
chrom 30 amino acid analyser (Biochrom, Cambridge,
UK).
Expression plasmids of glutathione
S-transferase -tagged proteins
The constructs expressing different variants of glutathione
S-transferase (GST)-PAGDTM have been described previ-
ously [22]. A construct expressing the SH3-domain of Fyn
was created by sub-cloning amino acids 83-537 into the
BamHI ⁄ NotI sites of the pGEX-4T-2 vector and intro-
ducing a stop codon at amino acid residue 142.
Biotin-peptide pull-down assay
Human peripheral blood T cells were purified from normal
donors by negative selection as described [18] and lysed in
ice-cold lysis buffer (50 mm Tris pH 7.4, 100 mm NaCl,
5mm NaF, 10 mm NaPP
i

,5mm EDTA, 1% Triton X-100,
50 mm n-b-octyl-d-glucoside, 1 mm Na
3
VO
4
and 1 mm
phenylmethylsulfonyl fluoride). After 30 min pre-clear at
4 °C using streptavidin beads (Invitrogen, Carlsbad, CA,
USA), T-cell lysates were incubated with different biotiny-
lated peptides for 3 h at 4 °C with rotation followed by
30 min incubation with streptavidin beads at room temper-
ature. Biotin alone was used as a control for non-specific
binding. Samples were washed extensively in ice-cold lysis
buffer prior to western blot analysis using anti-Fyn IgG
(Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).
Protein expression and purification
Recombinant proteins were expressed and purified as
described previously [22]. For thrombin cleavage of the
GST-tag, washed glutathione–Sepharose beads bound to
GST–Fyn SH3-domain were incubated with human throm-
bin (Sigma, St Louis, MO, USA) in 50 mm Tris (pH 8.0),
150 mm NaCl, 0.1% b-mercaptoethanol and 2.5 mm CaCl
2
at 4 °C overnight. The supernatant containing the Fyn SH3-
domain was purified further by gel filtration over a Superdex
75 column (GE Healthcare Europe GmbH, Uppsala,
Sweden) using 10 mm Hepes (pH 7.4), 150 mm NaCl, and
concentrated using a Vivaspin 2 spin column (Sartorius Bio-
lab, Auckland, New Zealand). Recombinant proteins were
stored at )80 °Cin10mm Hepes (pH 7.4), 150 mm NaCl.

In vitro kinase assay
Pulse-chase experiments using full-length, active Fyn kinase
(Upstate, Charlottesville, VA, USA) were performed as
described elsewhere [37] and protein phosphorylation quan-
tified using imagequant (GE Healthcare Europe).
Circular dichroism
CD spectroscopy was performed on a nitrogen-flushed
JASCO spectropolarimeter J810 (Jasco, Tokyo, Japan)
equipped with a circulating water bath. Samples were anal-
ysed at 20 °C using a quartz cuvette (Hellma GmbH & co,
Mu
¨
llheim, Germany) with a pathlength of 0.1 cm. CD spec-
tra were recorded five times at 50 nmÆmin
)1
with a width of
1 nm over a wavelength range of 190–260 nm. Samples
were analysed in 10 mm Hepes, pH 7.4, 150 mm NaCl.
Total buffer absorbance was analysed and was found to be
satisfactory for the range of wavelengths used in the
measurements. The scans were averaged and a sample-free
buffer spectrum was subtracted. Data smoothing was
S. A. Solheim et al. Modulation of the Fyn SH3–PAG interaction
FEBS Journal 275 (2008) 4863–4874 ª 2008 The Authors Journal compilation ª 2008 FEBS 4871
performed using an inverse square algorithm in sigmaplot
8.0 (SPSS, Chicago, IL, USA) as described previously [50].
Surface plasmon resonance
Measurements were performed on a BIAcore T100 instru-
ment (BIAcore Life Sciences ⁄ GE Healthcare Europe).
GST–Fyn SH3 fusion protein or GST protein alone

(diluted in 10 mm sodium acetate, pH 4.5) were immobi-
lized on CM5 sensor chips (BIAcore) via cross-linking of
free amine groups to the N-hydroxysuccinimide ⁄ 1-ethyl-3-
[3-dimethylaminopropyl] carbodiimide hydrochloride acti-
vated flow-cell surfaces to a final response of 8500 RU
(1 RU = 1 pgÆmm
)2
), followed by blocking of free succini-
mide ester groups with 1 m ethanolamine. GST–protein was
immobilised as a reference surface to the same molar
extent. The chip was treated with 10 mm dithiothreitol in
running buffer (10 mm Hepes, pH 7.4, 150 mm NaCl,
0.05% P20, 3 mm EDTA, 0.005% SDS) to remove GST
dimer products. After extensive washing of the surface with
running buffer, peptide binding was assessed by injecting
the indicated concentrations in running buffer over the
flow-cell surface at a sample rate of 5 lL Æ min
)1
. Kinetic
analysis was performed using the BIAcore T100 evaluation
software. Curve fitting was executed by the software pro-
gram (global fitting algorithm) which also performed calcu-
lations of dissociation constants (K
D
).
Molecular modelling of the protein–peptide
complexes
Modelling of the protein–peptide complexes was performed
using modeller [51] and visualized using pymol (http://
www.pymol.org). The Protein Data Bank [52] entry of the

Fyn tyrosine kinase SH3-domain bound to a proline-rich
peptide 3BP-2 (PPAYPPPPPVP, PDB ID: 1fyn [25]) and
P2L (PPRPLPVAPGSSKT, PDB ID: 1azg and 1a0n [26])
were used to establish the positioning of the PxxP core
motif of the peptides and 1azg was selected as a template
to create models for binding of the PRD1, PRD1-super,
and PRD1-RLP* peptides. In aligning the peptides of this
study to that in the 1azg, it was assumed that the core
SH3-domain binding motif (PxxP) would overlay with the
equivalent position as described in the original study [26].
Acknowledgements
This work was supported by Norwegian Research
Council FUGE Career Fellowship (159306 to TB), by
EU Grant 3D repertoire LSHG-CT-2005-212028 (to
RR), and by grants from the Norwegian Functional
Genomics Programme, the Norwegian Research
Council, the Norwegian Cancer Society, and the
European Union (grant no. 037189, thera-cAMP) (to
KT). We thank Gladys M. Tjørhom, Jorun Solheim,
and Ola Blingsmo for excellent technical assistance,
Dr Per E. Kristiansen for use of the circular dichro-
ism equipment, and Dr Matthew Betts for helpful
discussion.
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