High levels of structural disorder in scaffold proteins as
exemplified by a novel neuronal protein, CASK-interactive
protein1
Annama
´
ria Bala
´
zs
1,
*, Veronika Csizmok
2,
*, La
´
szlo
´
Buday
1,2
, Marianna Raka
´
cs
2
, Robert Kiss
3
,
Mo
´
nika Bokor
4
, Roopesh Udupa
2
,Ka

(Received 26 February 2009, revised 15
April 2009, accepted 12 May 2009)
doi:10.1111/j.1742-4658.2009.07090.x
CASK-interactive protein1 is a newly recognized post-synaptic density
protein in mammalian neurons. Although its N-terminal region contains
several well-known functional domains, its entire C-terminal proline-rich
region of 800 amino acids lacks detectable sequence homology to any
previously characterized protein. We used multiple techniques for the struc-
tural characterization of this region and its three fragments. By bioinfor-
matics predictions, CD spectroscopy, wide-line and
1
H-NMR spectroscopy,
limited proteolysis and gel filtration chromatography, we provided evidence
that the entire proline-rich region of CASK-interactive protein1 is intrinsi-
cally disordered. We also showed that the proline-rich region is biochemi-
cally functional, as it interacts with the adaptor protein Abl-interactor-2.
To extend the finding of a high level of disorder in this scaffold protein, we
collected 74 scaffold proteins (also including proteins denoted as anchor
and docking), and predicted their disorder by three different algorithms.
We found that a very high fraction (53.6% on average) of the residues fall
into local disorder and their ordered domains are connected by linker
regions which are mostly disordered (64.5% on average). Because of this
high frequency of disorder, the usual design of scaffold proteins of short
globular domains (86 amino acids on average) connected by longer linker
regions (140 amino acids on average) and the noted binding functions of
these regions in both CASK-interactive protein1 and the other proteins
studied, we suggest that structurally disordered regions prevail and play
key recognition roles in scaffold proteins.
Structured digital abstract
l

shown in this work, and is presumably involved
in the signal pathway related to the Abl tyrosine
kinases (A. Balazs, V. Csizmok, P. Tompa, R. Udupa
& L. Buday, unpublished results). The molecular
mechanism of the function of Caskins is not known at
Fig. 1. The diagram at the bottom shows a schematic representation of the domain structure of Caskin1. The N-terminal half contains six
ankyrin repeats, one SH3 domain and the two SAM domains, whereas the C-terminal half contains no recognizable domain, and has been
designated as a proline-rich region ⁄ domain (PRD). The proline-rich region was cut into three parts (PRD1, PRD2 and PRD3), cloned and char-
acterized individually in this work. Above the scheme is the prediction by the IUPred algorithm, which shows that the entire PRD region is
probably intrinsically disordered (the score is above 0.5).
l
MINT-7034579: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with ABI2 (uni-
protkb:
Q9NYB9)bytwo hybrid (MI:0018)
l
MINT-7034720: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Synapto-
tagmin (uniprotkb:
P21579)bytwo hybrid (MI:0018)
l
MINT-7034691: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Neurexin-2
(uniprotkb:
Q9P2S2)bytwo hybrid (MI:0018)
l
MINT-7034617: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with CASK
(uniprotkb:
P07498)bytwo hybrid (MI:0018)
l
MINT-7034748: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with SIAH1
(uniprotkb:
Q8IUQ4)bytwo hybrid (MI:0018)

multiple binding partners (without adherence to the
accepted definition of scaffolds) has been suggested
and analysed [2].
As a result of the rapid advance of knowledge
on intrinsically disordered ⁄ unstructured proteins
(IDPs ⁄ IUPs), the concept of protein disorder has
gained general recognition recently [3–6]. Physical
evidence exists for the disorder of about 500 proteins
[7], and bioinformatics predictions suggest that disor-
der is prevalent in the proteome of eukaryotes, with
more than 10% of their proteins being fully disordered
[8–10]. Disorder is most often implicated in signalling
and regulatory functions, and its functional benefits
often manifest themselves in protein–protein recogni-
tion [5,11]. One advantage often referred to is that
their extended structure enables IDPs to have a large
interaction capacity with small protein size [12], which
might be directly related to the involvement of disor-
der in scaffold proteins. In fact, there is an elevated
level of disorder in hub proteins, i.e. proteins involved
in multiple interactions [13–16], and disorder increases
with the number of proteins in multiprotein complexes
[17]. The functional role of structural disorder has
been noted in a few scaffold proteins, such as Sterile 5
(Ste5) [18], BRCA1 [19], CREB-binding protein (CBP)
[4] and Mypt1 [20], and it has been suggested that
flexibility provided by disorder is instrumental in
overcoming steric hindrance in the assembly of large
multiprotein complexes [21].
Motivated by the apparent relationship between pro-

formatics predictions by the IUPred algorithm
(Fig. 1). High IUPred scores indicate that the entire
proline-rich region of Caskin1 (amino acids 603–1430)
is disordered.
To confirm this prediction, a variety of experimental
approaches were also applied, as earlier it has been
suggested [5] that, as a result of the limitations of most
techniques, a multitude of approaches need to be
applied for the conclusive demonstration of disorder.
The full-length proline-rich region of Caskin1 with a
histidine tag on its C-terminus (PRD-His) was cloned
and expressed in bacteria. However, the expression of
this construct was rather difficult because of the high
proteolytic sensitivity of the protein, characteristic of
IDPs. Therefore, only CD, gel filtration and limited
proteolysis experiments could be performed, which do
not require large amounts of protein. For detailed
studies, the full-length proline-rich region was cut into
three parts, selected for splitting at sites of high local
disorder in the IUPred prediction (PRD1-His, Lys603–
Lys804; PRD2-His, Val805–Ala1199; PRD3-His,
Glu1200–Glu1430), cloned into PQE2 and pET20b
vectors with a C-terminal His tag and expressed in
Escherichia coli.
One important feature of IDPs is their heat stability.
Therefore, purification of the full-length proline-rich
region and its fragments from the bacterial extracts
was started by boiling the proteins at 100 °C for 5 min
and loading the supernatants on an Ni–agarose affinity
chromatograph. The heat stability of the fragments

of the proline-rich region, as the apparent molecular
mass (m
app
) of PRD-His (334.5 kDa) is 3.9 times
higher than the real value (85.9 kDa) (Fig. 2C). The
three fragments also show a high apparent molecular
mass: 4.5 (PRD1-His, 95.5 kDa), 2.2 (PRD2-His,
91.9 kDa) and 5.4 (PRD3-His, 125.4 kDa) times
higher than the real molecular mass (21, 41.7 and
23.2 kDa, respectively) (Fig. 3D). Because the column
was calibrated with globular proteins, these ratios sug-
gest a largely unfolded conformational state, as values
of m
app
⁄ m = 4–5 are typical of fully disordered
proteins [20].
We have demonstrated previously that the high
hydration potential of IDPs can be detected by wide-
line
1
H-NMR measurements [32,33]. This technique is
suitable for the measurement of the amount of bound
water after freezing out bulk water. We compared the
temperature dependence of the mobile water fractions
of the three fragments PRD1-His, PRD2-His and
PRD3-His (Fig. 3C). The amount of water in the
hydrate layer far exceeds that of BSA and approaches
that of ERD10, an IDP characterized previously [33],
which provides further evidence for the open and
largely solvent-exposed nature. It is of note that the

1
H chemical shifts are typical of IDPs [34].
The proline-rich regions of Caskin1 interact
with Abi2
To demonstrate that the proline-rich regions character-
ized above are biochemically functional, we studied the
interaction of Caskin1 fragments with Abi2, which is
an adaptor protein identified originally by its inter-
action with Abl tyrosine kinase [35]. Caskin1 was cut
into five regions and expressed as glutathione transfer-
ase (GST) fusion proteins. These protein regions repre-
sent the ankyrin repeats and the SH3 domain together
(ANK ⁄ SH3-GST), the two SAM domains (SAM-GST)
and the three proline-rich regions (PRD1–3-GST) of
the C-terminal PRD. The full-length PRD of Caskin1
was also expressed (PRD-GST). Green fluorescent pro-
tein (GFP)-tagged Abi2 was expressed in COS7 cells,
extracts of which were used for the GST pull-down
assay. As shown in Fig. 5, the first and second proline-
rich regions of Caskin1 (PRD1-GST and PRD2-GST)
A B
C D
Fig. 3. Structural characterization of fragments of PRD. (A) Far-UV CD spectra of PRD1-His (blue), PRD2-His (green) and PRD3-His (red). All
spectra show a characteristic minimum at around 200 nm, which underscores the unstructured nature of the proline-rich region. (B) Compar-
ison of the far-UV CD spectrum of the full-length PRD-His (full line) and the sum of the spectra of PRD1-His, PRD2-His and PRD3-His (bro-
ken line). The sum of the spectra of the three fragments reproduces the spectrum of PRD-His, which confirms the overall random structure
of the full-length proline-rich region and the lack of appreciable long-range structural organization within this region of the protein. (C) The
temperature dependence of the mobile water fraction of PRD1-His (blue), PRD2-His (green) and PRD3-His (red), compared with that of the
globular control BSA (cyan) and the disordered control ERD10 (black). The large amount of water in the hydrate layer of PRDs suggests their
open, solvent-exposed conformations. (D) Gel filtration chromatography of the fragments PRD1-His, PRD2-His and PRD3-His shows that all

important scaffold molecules modulating signalling
pathways at the post-synaptic sites of brain excitatory
synapses [36]. Ste5 serves in the yeast mating pathway,
ensuring that components of the mitogen-activated
protein kinase (MAPK) cascade, also involved in
osmoresponse and filamentation pathways, act specifi-
cally [18]. In our case, Caskin1 has been found in a
yeast two-hybrid screen to bind about 10 other part-
ners besides Abi2 (Table 1), and several points suggest
that it is a bona fide scaffold protein: (a) Caskin1 has
a modular structure with several of its domains and
non-domain regions involved in protein–protein inter-
actions; (b) none of its domains shows catalytic func-
tion; (c) it has 11 different partners all involved in
signal transduction; (d) it is preferentially located in
the PSD, known to harbour many proteins of signal-
ling and scaffold function (e.g. PSD95, Shank, Homer,
etc. [37]); (e) it has long uncharacterized regions which
lack sequence similarity to other proteins, and has
been shown here to be intrinsically disordered. The
appearance and functional role of structural disorder
have been explicitly noted in other scaffold proteins,
such as Ste5 [18], BRCA1 [19], CBP [4] and Mypt1
[20]. Thus, we decided to study this feature in detail to
gain further insight into the possible importance of
disorder in Caskin1 function and the class of scaffolds
in general.
The collection of scaffold proteins for bioinformatics
study, however, is hampered by the lack of consensus
on the definition of these proteins. In this article, we

homology domain, a myristoylation site or a short
transmembrane domain. After direct or indirect inter-
actions with a tyrosine kinase, the docking protein
becomes tyrosine phosphorylated on multiple sites that
can interact with signalling proteins containing SH2
domains. Insulin receptor substrate 1, for example, con-
tains an N-terminal Pleckstrin homology domain and a
phosphotyrosine-binding domain, and nearly 20 poten-
tial tyrosine phosphorylation sites at the C-terminus
[39]. As suggested above, scaffold proteins are able to
interact with many different proteins at the same time,
but they are typically not subject to phosphorylation,
which creates novel binding sites. The lack of consensus
on these definitions is also indicated by the sole study
addressing the structural disorder in scaffold proteins
[2], in which several proteins clearly not of scaffold
function (e.g. p53, a transcription factor and voltage-
activated potassium channel, a binding partner of the
scaffold protein PSD95 [40]) were involved. Our study
encompasses proteins involved in the formation of mul-
tiprotein complexes, which have modular organization.
We collected 74 such proteins by literature search and
analysed their disorder by three different algorithms.
Prediction of disorder in scaffold proteins
The structural disorder of the 74 scaffold, docking and
anchor proteins was predicted by three different algo-
rithms, i.e. IUPred, VSL2 and FoldIndex (Table S1,
see Supporting information). We found that the ratio
of residues in local disorder was very high (49.7%,
63.36% and 47.82% predicted by IUPred, VSL2 and

proximity is rather difficult in the case of a rigid, glob-
ular structure, it is reasonable to assume that these
proteins contain long, disordered regions. Nevertheless,
the occurrence of disorder and its functional conse-
quences in scaffold proteins have never been examined
systematically. The present study provides evidence for
the extensive disorder of Caskin1 and also for the class
of scaffold proteins in general. Overall, the level of dis-
order exceeds that of the functional class so far consid-
ered to be the most disordered: RNA chaperones [27].
It is known that, in proteins associated with signal
transduction, transcription and RNA chaperone activi-
ties, the ratio of amino acids in locally disordered
regions is very high, on the order of 50–60%. These high
levels are thought to result from the functional advanta-
ges provided by disorder, which enables functions that
cannot be carried out by globular proteins. One advan-
tage of the extended, disordered conformation is an
Table 1. Results from the two-hybrid screen using a fragment of
Caskin1 (amino acids 280–963) as bait and a human fetal cDNA
library. The numbers in parentheses represent the number of identi-
cal clones obtained.
Clone Protein Function
1 (12) Abl-interactor-2 (Abi2) Adaptor protein
2 (2) CASK Scaffold protein
3 (1) EphA2 Receptor tyrosine kinase
4 (1) L1CAM Cell adhesion molecule
5 (2) Myosin IB Class I myosin
6 (1) Nck1 Adaptor protein
7 (1) Neurexin 2 Neuronal cell adhesion

region has never been examined. According to our
structural studies, this entire region is intrinsically dis-
ordered, and proline-rich regions are known to interact
with SH3, WW and other domains of cognate proteins
[31,44]. Indeed, PRD of Caskin1 contains several
consensus SH3 binding sites, and we postulate that it
is involved in multiple interactions with other PSD
proteins. In this study, we have shown that the
proline-rich regions interact with the Abi2 protein,
which have SH3 domains (we have also found the
in vivo association of Abi2 with Caskin1; A. Balazs,
V. Csizmok, P. Tompa, R. Udupa & L. Buday,
unpublished results). In this sense, PRD of Caskin1
might function in a manner similar to the long, central,
disordered region of BRCA1, which harbours binding
motifs for multiple partners in DNA repair [19]. A fur-
ther point on the function of PRD of Caskin1 is that
all of our studies point to a local tendency of ordering
in the middle PRD2 segment (amino acids 805–1199).
The level of hydration of this fragment is lower than
that of the other two and the results of CD analysis
also show some deviation from a fully disordered,
random coil-like state. By gel filtration chromatogra-
phy, this region also shows less extended conformation
than the rest of PRD. As a local tendency for ordering
is a sign of sites poised for interactions [45,46], it
Fig. 6. Schematic representation of the domain structure of selected scaffold proteins. The scheme shows the domain architecture of 20
selected scaffold proteins representing 20 families described in detail in the literature established by PFAM. Long grey lines connecting the
domains are regions with no recognizable similarity to known proteins.
Fig. 7. Length distribution of domains and linker regions in scaffold

collection of 74 scaffold proteins: on average, 53.6%
of their amino acids were in locally disordered regions.
Disorder, however, is not evenly distributed in the
sequences, as shown by the consideration of only the
regions connecting PFAM domains. The predicted
average disorder for these regions is 64.5%, which sug-
gests that scaffold proteins are constructed as beads on
a string from globular domains connected by occasion-
ally very long linker regions. Because these linkers
cover 65.8% of the total length of scaffold proteins on
average, and their average length far exceeds that of
the globular domains, there is no doubt that disorder
in these proteins fulfils very important functions, prob-
ably commensurable in importance with that of
ordered domains.
Actual data on some scaffold proteins provide
evidence that these regions are much more than mere
passive linkers of functional globular domains. For
example, BRCA1 contains an approximately 1500-
amino-acid-long central region between the N-terminal
RING domain and C-terminal BRCT domain [19].
Although it lacks stable structural elements or recog-
nizable domains, this region is implicated in binding
not only DNA, but numerous proteins involved in
DNA damage response and repair [49,50]. Another
scaffold protein, Mypt1, also contains a long disor-
dered segment in its N-terminal region, and this
segment is involved in binding to the type 1 protein
phosphatase [51]. CBP has also been amply character-
ized in this respect. This protein contains seven globu-

DNA constructs
The full-length rat Caskin1 cDNA was kindly provided by
Thomas Su
¨
dhof (University of Texas Southwestern Medical
Center, Dallas, TX, USA), and the full-length Abi2 cDNA
was donated by Ann Marie Pendergast (Duke University
Medical Center, Durham, NC, USA). Caskin1 cDNA was
amplified by a high-fidelity DNA polymerase and subcloned
into the pcDNA 3.1 ⁄ V5-His TOPO vector (Invitrogen, San
Diego, CA, USA). The full-length Abi2 was amplified by
PCR and subcloned into the BamHI site of the pEGFP-C1
vector (BD Biosciences Clontech, San Jose, CA, USA).
cDNAs corresponding to the ankyrin repeats and SH3
domain (ANK ⁄ SH3-GST, amino acids 1–346), SAM
domains (SAM-GST, amino acids 347–610), proline-rich
region 1 (PRD1-GST, amino acids 603–804), proline-rich
region 2 (PRD2-GST, amino acids 804–1199), proline-rich
region 3 (PRD3-GST, amino acids 1200–1430) and the
full-length PRD of Caskin1 (PRD-GST, amino acids
603–1430) were amplified by PCR and subcloned into the
EcoRI ⁄ SalI sites of the pGEX-4T1 vector (Amersham
Biosciences, Fairfield, CT, USA) as GST fusion proteins.
High levels of disorder in scaffold proteins V. Csizmok et al.
3752 FEBS Journal 276 (2009) 3744–3756 ª 2009 The Authors Journal compilation ª 2009 FEBS
For pull-down experiments, GST fusion proteins were puri-
fied by binding to glutathione–agarose (Sigma, St Louis,
MO, USA) without elution. Protein purification was moni-
tored on Coomassie blue-stained SDS–PAGE gels: the
majority of the GST proteins gave single bands.

)1
in 10 mm Na
2
HPO
4
, 150 mm NaCl, pH 7.5 in
a cuvette (path length, 1 mm) on a Jasco J-720 spectropola-
rimeter (Jasco, Oklahoma City, OK, USA) in a continuous
mode with a bandwidth of 1 nm, response time of 8 s and
scan speed of 20 nmÆmin
)1
. All spectra shown were
obtained by subtracting the buffer spectrum and averaging
10 separate scans.
Gel filtration chromatography
The unfolded nature of PRD and its fragments was also
characterized by gel filtration chromatography. The
proteins (200 lL) were run on an Amersham Biosciences
Superdex 200 (1 · 30 cm) column at 0.5 mLÆmin
)1
in a
buffer of 50 mm Na
2
HPO
4
, 150 mm NaCl, pH 7.0 on an
Amersham Biosciences FPLC system. The proteins were
detected at 280 nm. The column was calibrated using the
following globular proteins (m in parentheses): ribonuclease
A (13.7 kDa), chymotrypsinogen A (25.0 kDa), ovalbumin

ple is in the liquid state. Details of the applied method have
been described elsewhere [32,33,55].
The effect of freezing on protein solutions was controlled
by the comparison of NMR parameters obtained before
and after a freeze–thaw cycle at temperatures above 0 °C.
We found that the freeze–thaw cycle caused no observable
changes for the studied samples as far as the measured
NMR parameters were concerned. The temperature was
controlled by an open-cycle Oxford cryostat with a stability
of ± 0.1 °C; the uncertainty of the temperature scale was
±1°C.
1
H-NMR measurements and data acquisition were
accomplished using a Bruker SXP 4-100 NMR pulse spec-
trometer at x
0
⁄ 2p = 82.55 MHz with a stability of better
than ± 10
)6
. The data points in the figures are based on
spectra recorded by averaging signals to reach a signal to
noise ratio of 50. The number of averaged NMR signals
was varied to achieve the desired signal quantity for each
sample and for unfrozen water quantities. The sensitivity of
the NMR spectroscope on sample change was controlled
by measuring the length of the p ⁄ 2 pulse to obtain reliable
M
0
values [55]. The extrapolation to zero time was
performed by fitting a stretched exponential.

EGTA, 1 mm Na
3
VO
4
,1mm p-nitrophenylphosphate,
10 mm benzamidine, 1 mm phenylmethylsulphonyl fluoride
and 25 lg ÆmL
)1
each of leupeptin, soybean trypsin inhibitor
and aprotinin. The lysates were clarified by centrifugation
at 15 000 g for 10 min at 4 °C. The lysates were then pre-
cipitated with 20 lg of the indicated GST-fusion protein
immobilized on glutathione–agarose (Sigma) for 1 h at
4 °C. Protein precipitates were washed three times with ice-
cold NaCl ⁄ P
i
, pH 7.4, containing 0.4% Triton X-100 and
eluted with SDS sample buffer. Bound proteins were sepa-
rated by SDS-PAGE and, because of the small amount of
proteins, transferred to nitrocellulose membrane and immu-
noblotted with the indicated antibodies. Blots were devel-
oped by the enhanced chemiluminescence (ECL; Amersham
Biosciences) system.
Collection of scaffold proteins and bioinformatics
predictions
We collected a number of anchor, docking and scaffold pro-
teins (denoted collectively as scaffold proteins) from the litera-
ture and by screening the UniProt knowledgebase (Table S1).
For the prediction of disorder, three different algorithms,
i.e. IUPred [13,23], VSL2 [24,25] and FoldIndex [26], were

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Supporting information
The following supplementary material is available:
Fig. S1. SDS-PAGE with the various forms of Caskin1