Intrinsic disorder in the C-terminal domain of the
Shaker voltage-activated K?channel modulates
its interaction with scaffold proteins
Elhanan Magidovich*†, Irit Orr*, Deborah Fass‡, Uri Abdu*, and Ofer Yifrach*†§
*Department of Life Sciences and†Zlotowski Center for Neurosciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel;
and‡Department of Structural Biology, Weizmann Institute of Science, Rehovot 76100, Israel
Edited by Ramo ´n Latorre, Centro de Estudios Cientı ´ficos, Valdivia, Chile, and approved June 26, 2007 (received for review May 2, 2007)
The interaction of membrane-embedded voltage-activated po-
tassium channels (Kv) with intracellular scaffold proteins, such
as the postsynaptic density 95 (PSD-95) protein, is mediated by
the channel C-terminal segment. This interaction underlies Kv
channel clustering at unique membrane sites and is important
for the proper assembly and functioning of the synapse. In the
current study, we address the molecular mechanism underlying
Kv/PSD-95 interaction. We provide experimental evidence,
based on hydrodynamic and spectroscopic analyses, indicating
that the isolated C-terminal segment of the archetypical Shaker
Kv channel (ShB-C) is a random coil, suggesting that ShB-C
belongs to the recently defined class of intrinsically disordered
proteins. We show that isolated ShB-C is still able to bind its
scaffold protein partner and support protein clustering in vivo,
indicating that unfoldedness is compatible with ShB-C activity.
Pulldown experiments involving C-terminal chains differing in
flexibility or length further demonstrate that intrinsic disorder
in the C-terminal segment of the Shaker channel modulates its
interaction with the PSD-95 protein. Our results thus suggest
that the C-terminal domain of the Shaker Kv channel behaves as
an entropic chain and support a ‘‘fishing rod’’ molecular mech-
anism for Kv channel binding to scaffold proteins. The impor-
tance of intrinsically disordered protein segments to the com-
plex processes of synapse assembly, maintenance, and function
channel clustering ? intrinsically disordered ? PSD-95
tions between closed and open states in response to changes in
membrane potential (1–3). This form of gating underlies many
fundamental biological processes, in particular the generation of
nerve and muscle action potentials (4). The involvement of Kv
channels in shaping action potentials is primarily based on the tight
interaction between the channel’s voltage-sensing and pore do-
mains (4). Effective transmission of the action potential to a target
cell across the synaptic cleft requires precise channel localization
of certain Kv channels (7, 8). It has been demonstrated that a
conserved PDZ-binding motif present at the end of the C-terminal
segment of the prototypical Shaker Kv channel is an important
determinant for binding to the Drosophila postsynaptic density 95
(PSD-95) scaffold protein, Dlg, a member of the membrane-
associated guanylate kinase family (5, 6, 9). This interaction is
and has been implicated in synaptic growth and plasticity (5, 6).
The molecular mechanism underlying the interaction of PSD-95
and Kv channels is not yet clear. Previous studies highlighted the
role played by the 4-aa-long C-terminal PDZ-binding motif of the
channel while ignoring the long stretch of residues to which this
motif is attached. Recently, however, we have presented bioinfor-
matics evidence to argue that intrinsic disorder at the C-terminal
tail segments of Kv channels preceding the PDZ-binding motif is
oltage-activated potassium channels (Kv) are modular mem-
brane-spanning proteins that undergo conformational transi-
another important determinant for channel binding to the PSD-95
scaffold protein partner (10). This conclusion was based on the
unusual sequence characteristics of the C-terminal segments of
many Kv channel members. Inspection of the C-terminal sequence
of the Shaker Kv channel (sw: P08510), for example, reveals an
enrichment in hydrophilic amino acids, depletion of hydrophobic
usage in the Shaker C-terminal sequence reveals a depletion in
order-promoting amino acids (i.e., W, C, F, I, Y, V, and L) and an
enrichment in disorder-promoting residues (i.e., S, A, P, R, E, K,
and Q), relative to the overall amino acid usage in the entire
Drosophila melanogaster proteome (Fig. 1A). Such sequence char-
acteristics, together with consideration of mean net charge and
mean hydrophobicity values, explains why the C-terminal segments
of both the Shaker and rat Kv 1.2 channels, for instance, are
expected to belong to the recently defined intrinsically disordered
protein family (Fig. 1B) (11). Protein segments belonging to this
group bear sequence characteristics that oppose folding and are,
therefore, unstructured or intrinsically disordered under physiolog-
ical conditions (12–17). The lack of or ambiguity regarding struc-
ture at the C-terminal segments of the Kv 1.2 and Shaker channels,
as revealed by x-ray crystallography (18) and cryo-EM (19) anal-
yses, respectively, lends credence to this conclusion.
To understand the functional advantages of an intrinsically
disordered C-terminal segment for the Kv channel protein, phylo-
genetic inference analysis of the Kv channel family was conducted
(10). Such analysis provided evidence that, throughout evolution,
the appearance of intrinsic disorder at the channel’s C-terminal tail
is associated with the presence of the PDZ-binding motif in the
same part of the protein, implying that both tail region traits are
important for PSD-95 scaffold protein binding (10). Based on this
assertion, an intermolecular ‘‘fishing rod’’ mechanism for Kv chan-
nel binding to scaffold proteins was proposed (Fig. 1C) (10) in
which the membrane-embedded Kv channel is anchored at unique
membrane sites by binding to a scaffold protein using a C-terminal
sequence that contains a flexible string (an intrinsically disordered
chain) and a hook (the PDZ-binding motif) at its tip. Disorder at
the channel C terminus would provide the orientational freedom
needed for searching and successfully connecting to the PSD-95
scaffold protein partner.
In the present report, we have experimentally addressed predic-
tions emerging from sequence and phylogenetic analyses to assess
Author contributions: O.Y. designed research; E.M. and I.O. performed research; D.F. and
U.A. contributed new reagents/analytic tools; E.M. and O.Y. analyzed data; and D.F. and
O.Y. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviation: Kv, voltage-activated potassium channels.
§To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
August 7, 2007 ?
vol. 104 ?
no. 32 www.pnas.org?cgi?doi?10.1073?pnas.0704059104
the feasibility of the fishing rod mechanism for Kv channel clus-
tering proposed above. We demonstrate that the isolated C-
terminal domain of the Shaker channel is indeed intrinsically
and channel portion of the protein. Furthermore, we demonstrate
that the intrinsically disordered nature of the Shaker channel
C-terminal tail modulates its interaction with the PSD-95 scaffold
protein, thus supporting the fishing rod mechanism of Kv channel
clustering by PSD-95.
The C-Terminal Domain of the Shaker K?Channel Is Intrinsically
Disordered. Previous structural analysis of the Kv 1.2 channel by
x-ray crystallography (18) showed no electron density correspond-
ing to the C-terminal region. Difference density calculations from
2D averages and 3D reconstructions derived from electron micros-
copy images of full-length Shaker channels vs. a C-terminally
truncated mutant (19) indicated a region that could correspond to
all or part of the C-terminal segment, although the mass associated
with this region could not be reliably determined. Taken together,
these results are consistent with a lack of ordered structure or a
to the membrane-spanning portion.
To experimentally assess the structural state of the Shaker
channel C-terminal segment, ShB-C, one must first determine an
appropriate N-terminal boundary for this region. Boundaries of
long intrinsically disordered loops inserted into structural domains
were shown to correlate with the boundaries of exons in the
encoding gene (20). Although ShB-C corresponds to an extrado-
main segment and not an intradomain loop, we examined the
Shaker channel gene structure and noted that the codon encoding
Val-513 marked the start of the exon following the sixth transmem-
brane helix of the protein. This objective assignment of the N-
terminal boundary of ShB-C proved useful, as protein constructs
starting upstream of Val-513 aggregated during purification (not
We next cloned, expressed, and purified the C-terminal domain
of the Shaker channel [see supporting information (SI) Text]
starting from Val-513 and examined, using hydrodynamic and
spectroscopic approaches, its structural state when isolated from
the rest of the channel and the membrane. ShB-C migrates in a
size-exclusion column unusually fast for a 15.9-kDa protein (Fig.
2A). Compared with the 19.7-Å Stokes radius expected for a
compact globular protein of this size (21), a Stokes radius of 32.9
Å was estimated for ShB-C, based on a calibration curve created
using standard proteins of known molecular weight (Fig. 2B). This
anomaly could be explained either by ShB-C oligomerization or by
an expanded or elongated monomeric state of this domain. To
discriminate between the two possibilities, analytical ultracentrifu-
gation was performed. The data obtained were consistent with a
monodispersed population of protein with molecular mass of 15.5
kDa, in good agreement with the monomer molecular mass calcu-
lated from the protein sequence (15,916 Da), confirmed by mass
spectrometry. The gel filtration and analytical ultracentrifugation
results can be reconciled by calculation of the expected Stokes
radius for an intrinsically disordered monomeric protein of 15.9
kDa. The calculation, which yielded a value of 33.1 Å, was per-
formed according to Uversky (21), who observed that the experi-
mentally measured Stokes radii of known random coil proteins
is described by: logRs(coil) ? ?0.551 ? 0.493 ? logM, where
Rs(coil) is the expected Stokes radius for a random coil chain, and
scattering further supports this conclusion, yielding a Stokes radius
of 37.2 Å (data not shown; see SI Text for further details).
CD is a sensitive spectroscopic method for analyzing the
presence or absence of secondary structural elements, in par-
ticular ?-helices, within a protein. The far-UV CD spectrum for
ShB-C (Fig. 3A) lacked the typical signatures of secondary
structure. Neither negative peaks at 222 and 208 nm, typical for
?-helices (Fig. 3A, see ovalbumin trace) nor the negative peak at
?217 nm, typical for ?-sheets, were observed in the CD spec-
trum of ShB-C. Instead, ShB-C exhibited a negative peak at 200
nm, indicative of a strong contribution from disordered struc-
tural elements, characteristic of a protein in a random coil
conformation. Although ShB-C contains eight aromatic resi-
dues, its CD spectrum in the near-UV range (250–330 nm) was
devoid of any ellipticity (not shown), indicating the absence of
oriented aromatic residues typically present in the hydrophobic
cores of compact globular proteins. The ellipticity of ShB-C at
222 nm did not change appreciably with increasing temperature
up to 100°C and failed to reveal any cooperative transition (Fig.
3B). The slight negative slope of the signal as a function of
temperature is further typical of random coil proteins. Although
changes in environmental conditions can influence the distri-
promoting amino acid content across the entire Drosophila proteome (black
bars) and in ShB-C (gray bars). (B) The mean net charge and mean hydropho-
bicity values of the Shaker and Kv 1.2 channels (black circles) lie within the
solid line is an empirical linear regression that defines the boundary between
folded (black squares) and intrinsically disordered (gray circles) proteins
(adapted with permission from ref. 11). (C) A fishing rod mechanism for Kv
channel binding to scaffold proteins. A voltage-gated K?channel interacts
with the PSD-95 scaffold protein upon binding of the C-terminal PDZ-binding
shape, box, and rectangular shapes represent the PDZ, SH3, and guanylate
kinase domains of the PSD-95 protein, respectively.
The C-terminal tail domain of the Shaker Kv channel is predicted to
Magidovich et al.
August 7, 2007 ?
vol. 104 ?
no. 32 ?
bution of conformations of intrinsically disordered proteins (21),
the random coil signature of ShB-C, as revealed by CD analysis,
was not altered over a wide range of pH values (3–12), salt
concentrations (20–300 mM), or different concentrations of
SDS or trifluoroethanol (not shown). The CD results indicate
that ShB-C does not possess significant secondary structure and
suggest a lack of fixed tertiary structure as well.
Analysis of ShB-C by NMR provides unequivocal evidence for
the lack of tertiary structure for this protein segment. The varied
chemical environments experienced by protons of a folded protein
result in a complex NMR spectrum with many nonoverlapping
peaks dispersed over a wide range of chemical shifts. When,
however, a protein is found to lack a fixed conformation, any given
proton is under the influence of only those atoms to which it is
bonded, thereby giving rise to a spectrum similar to that of
noninteracting amino acids. Such a spectrum represents a summa-
tion of the NMR spectra of individual amino acids and will appear
peaks. Accordingly, the1H NMR spectrum of ShB-C (SI Fig. 6A)
lacks the chemical shift dispersion typical of folded proteins, as
exhibited by the 9-kDa snake venom toxin, ?-bungarotoxin (22),
shown for comparison (SI Fig. 6B). Rather, the profile of ShB-C
resembles the1H NMR spectrum of the C-terminal domain of
(23). Hence, our results suggest that the C-terminal portion of the
Shaker channel is an intrinsically disordered random chain.
The Isolated Shaker Channel Tail Domain Interacts with Its PSD-95
Scaffold Protein Partner to Mediate Protein Clustering in Vivo. We
next examined whether the intrinsically disordered isolated ShB-C,
out of its membrane and channel contexts, is capable of binding its
PSD-95 scaffold protein partner, and whether this interaction can
mediate protein clustering in vivo. For this purpose, we transfected
the Drosophila Schneider cell line with DNA encoding a PSD-95-
GFP fusion protein and conducted pulldown assays using ShB-C as
bait, as schematically depicted in Fig. 4A. Such an experimental
design mimics the spatial constraint of membrane-integrated
ShB-C, allowing us to probe the interaction of this Kv channel
segment with PSD-95. The results reveal that His6-tagged ShB-C,
immobilized on Ni2?beads via its N terminus, is able to capture
PSD-95 from a crude soluble protein extract of transformed
Drosophila Schneider cells (Fig. 4B), leading us to conclude that
ShB-C retains its function in this protocol. To assess whether the
intrinsically disordered chain of ShB-C can support protein clus-
tering in vivo, we next transfected Drosophila Schneider cells to
express PSD-95-GFP and ShB-C fused to the CD8 membrane
protein (6), either together or separately, and evaluated PSD-95
membrane association and ShB-C-CD8 clustering by confocal light
microscopy (see SI Text). As presented in Fig. 4C, transfection with
ness. (A) Size-exclusion chromatography elution profiles of ShB-C and chymo-
trypsinogen A, monitored at 280 nm. (B) Mobility [(?logKav)1/2; see Materials
and Methods] vs. Stokes radius plot, used for Stokes radius determination of
ShB-C, based on analytical size-exclusion chromatography of standard mono-
meric molecular weight markers. The open circle corresponds to ShB-C. The
black circles correspond to molecular weight markers, as indicated. (C) Equi-
librium sedimentation of ShB-C at 5°C and 19,000 rpm. Representative data
are plotted as ln (absorbance) against the square of the radius from the axis
of rotation. The slope is proportional to the molecular mass (see SI Text).
Dashed lines with increasing slopes indicate calculated values for monomeric
ShB-C (1), dimeric (2), trimeric (3), and tetrameric (4) forms of ShB-C. The data
are consistent with a monomeric model for ShB-C, as judged by the residuals
Hydrodynamic analyses reveal an ShB-C structure with low compact-
ShB-C. (A) Comparison of the far-UV CD spectra of ovalbumin and ShB-C (0.5
mg/ml), obtained at room temperature (25°C). The CD spectrum of ShB-C was
essentially similar at 90°C (not shown). (B) Temperature dependence of the
molar ellipticity of ShB-C (0.125 mg/ml), followed at 222 nm.
CD spectroscopic analysis reveals an extended conformation for
www.pnas.org?cgi?doi?10.1073?pnas.0704059104Magidovich et al.
DNA encoding the PSD-95-GFP fusion construct alone resulted in
homogeneous protein expression throughout the cytoplasm (Fig.
4C Upper Left). In the case of cells expressing the CD8-ShB-C
chimera alone, homogeneous protein expression was observed
stained red fluorescence pattern (Fig. 4C Upper Center). Cotrans-
fection with constructs encoding the PSD-95-GFP chimera and the
CD8 protein by itself yielded no change from the homogeneous
cytoplasmic green fluorescence pattern obtained with PSD-95-
GFP alone (Fig. 4C Upper Right). By contrast, cotransfection with
both the PSD-95-GFP- and CD8-ShB-C-encoding constructs re-
sulted in PSD-95 membrane association, as reflected in the green
(Fig. 4C Lower Left). This time, however, the red CD8-ShB-C-
derived fluorescence pattern, obtained using anti-CD8 antibodies
and a suitable wavelength of detection, revealed a nonhomoge-
neous membrane distribution of CD8-ShB-C (Fig. 4C Lower Cen-
ter). These discrete membrane domains of enhanced fluorescence
intensity may reflect some degree of protein clustering. Indeed,
examination of the membrane-associated yellow coloring of the
merged cell images (Fig. 4C Lower Right) clearly shows overlap
between the membrane association of PSD-95 and the redistribu-
tion of CD8-ShB-C in the presence of this scaffolding protein. The
ability of the Shaker channel C-terminal tail to support protein
clustering mediated by the PSD-95 protein was, furthermore,
demonstrated at the whole organism level (6). As such, we suggest
that ShB-C represents a functional module of the intact channel,
is compatible with its biological function in the context of the intact
Intrinsic Disorder at the Shaker Channel Tail Domain Regulates Its
Interaction with PSD-95. To what extent does the intrinsically
disordered nature of ShB-C regulate its interaction with the
PSD-95 scaffold protein? To address this question, we exploited
the pulldown experimental setup described above to qualita-
tively evaluate the interaction between the PSD-95 scaffold
protein and various versions of ShB-C exhibiting different
flexibilities or chain lengths. To achieve fast readout in our
pulldown assays, we challenged the anchored stationary ShB-C
protein or its mutants with a mobile phase containing not
full-length PSD-95 but rather its purified tandem PDZ-1 and -2
domains (PDZ12), previously shown to directly interact with the
Kv channel C-terminal tail (24). The use of this experimental
design to assess the interaction of the Kv channel C-terminal tail
with PSD-95, despite presenting a simplified view of the process,
is, nonetheless, informative. Capture of the purified PDZ12
protein (21.5 kDa) by bead-fixed ShB-C can be evaluated by
SDS/PAGE analysis with high sensitivity, as reflected in Fig. 5A
(lane 2), where the ability of full-length ShB-C protein to capture
significant amounts of PDZ12is shown. By contrast, as expected
from the results of previous studies (5–7), no interaction oc-
curred when an ShB-C mutant protein lacking its C-terminal
PDZ-binding motif was used as bait (compare lanes 2 and 3). A
significantly higher amount of PDZ12protein was trapped using
an ShB-C mutant protein lacking the entire intrinsically disor-
dered chain except for the last 11 C-terminal residues, which
include the PDZ-binding motif (compare lanes 2 and 4). To next
test whether flexibility of the Shaker C-terminal tail affected its
interaction with PSD-95, we performed a pulldown experiment
in which the entire intrinsically disordered sequence of ShB-C,
motif, was replaced by a globular cellulose-binding domain
(CBD) (see SI Text). The folded 17-kDa CBD moiety fused to
the Shaker channel PDZ-binding motif captured PDZ12 to a
greater extent than did the similarly sized but intrinsically
unstructured full-length ShB-C (compare lanes 2 and 5). When,
however, the intrinsically disordered C-terminal domain of
gliotactin (Gli-C), a nonrelated cell adhesion protein (23) iden-
tical in length but envisaged by all disorder predictors to be more
flexible, was fused to the Shaker channel PDZ-binding motif,
capture of PDZ12was achieved to a much lower extent (lane 6)
(refer to SI Text for calculation of disorder tendencies of ShB-C
and Gli-C). Differences in the amount of captured PDZ12by
each construct (Fig. 5B), relative to ShB-C, were all found to be
statistically significant (P ? 0.0001). These observed differences
cannot be attributed to changes in long-range electrostatic
interactions, because the amount of PDZ12 captured by the
various ShB-C-modified chains was not affected by changes in
ionic strength over a wide range of salt concentrations (50–300
mM; data not shown). Our binding experiments thus demon-
strate that, relative to ShB-C, shorter or less flexible protein
chains have higher affinity for the PDZ domains of the PSD-95
protein than do longer or more flexible chains. Assuming that
the intrinsically disordered chain of ShB-C does not fold upon
binding to PDZ12(see SI Appendix), these observations can be
explained by considering the contribution of entropy to binding.
Long chains, sampling more conformational states, lose more
entropy than do shorter or rigid structures upon association,
because the configurational space of the longer chains is re-
stricted to a greater degree. Taken together, our findings imply
that, in addition to its PDZ-binding motif, the intrinsically
for the batch pulldown assay. Ni2?-NTA bead-bound ShB-C protein containing the PDZ-binding motif at its C terminus (gray rectangular box) served as bait for
the capture of the modular PSD-95 partner protein. (B) SDS/PAGE (Left) and Western blot (Right) analyses of eluted fractions of a pulldown experiment
demonstrating the molecular interaction between ShB-C and PSD-95. Left lane corresponds to protein molecular weight markers. Lane 1 corresponds to a crude
with ShB-C-free or -bound beads, respectively. Western blot analysis of the same gel shown (Right) was performed by using anti-GFP primary antibodies (see SI
Text). (C) Confocal microscopic analysis of Drosophila S2 Schneider cells expressing PSD-95-GFP and/or ShB-C fused to the CD8 membrane-targeting sequence,
either separately (Upper) or together (Lower).
Isolated ShB-C binds its PSD-95 scaffold protein partner and supports protein clustering in vivo. (A) Schematic depiction of the experimental setup used
Magidovich et al.
August 7, 2007 ?
vol. 104 ?
no. 32 ?
disordered nature of the Shaker channel C-terminal tail preced-
ing this motif also acts as an important determinant in mediating
the binding of the channel to its PSD-95 scaffold protein partner.
Synaptic transmission in neuronal synapses requires the proper
organization of ion channels, neurotransmitter receptors, cell
adhesion proteins, cytoskeleton proteins, and signaling mole-
cules at the synaptic junction (25). Essential for synapse orga-
nization are multidomain scaffold proteins, like the PSD-95
protein, that interact with several different proteins, thereby
serving as nuclei for assembly of those macromolecular com-
plexes underlying synaptic architecture (26–30). Through their
PDZ domains, scaffold proteins anchor and cluster ion channels
at specific subcellular locations by interacting with one or more
consensus PDZ-binding sequence motifs located at the intracel-
lular C-terminal tails of the channels (28–30). Insight into the
molecular mechanism underlying the interaction of ion channels
with scaffold proteins may come from consideration of the
specific interaction between the Kv channel and PSD-95. Based
on bioinformatics sequence and phylogenetic inference analyses
of the Kv channel family, we recently suggested a fishing rod
mechanism to describe Kv channel binding to scaffold proteins
(10). In this model, the voltage-gated K?channel interacts with
PDZ domain(s) of the PSD-95 protein through binding of the
channel C-terminal PDZ-binding motif ‘‘hook,’’ tethered to the
rest of the channel protein by an extended chain.
For the fishing rod mechanism to hold true, two criteria must be
met. One must first demonstrate that the C-terminal domain of the
results presented in the current report provide evidence that the
C-terminal tail of the Shaker Kv channel is indeed intrinsically
disordered. The hydrodynamic properties of a polypeptide corre-
size exclusion chromatography, dynamic light scattering, and ana-
random coil protein (Fig. 2). Far-UV CD and NMR spectroscopic
analyses further indicated that ShB-C lacks secondary and tertiary
structure (Fig. 3 and SI Fig. 6). Moreover, we have shown that the
isolated intrinsically disordered ShB-C protein, out of its native
channel and membrane contexts, remains functional, maintaining
pool of Drosophila Schneider cell proteins and supporting protein
clustering in vivo in the same cell line (Fig. 4). A lack of intrinsic
structure may, therefore, be inherent to the C-terminal tail of the
of the PDZ-binding motif (the possibility that ShB-C may acquire
subunits is discussed in SI Appendix).
Our results further provide evidence that the intrinsically disor-
dered nature of the C-terminal chain modulates the interaction of
the Kv channel with the PSD-95 scaffold protein, the second
requirement for proof that the fishing rod model accurately de-
scribes the mechanism of Kv channel binding to scaffold proteins.
Assuming a simple one-step binding reaction of the random coil
chain of ShB-C to PSD-95 and considering the entropic contribu-
tion of the binding reaction, shorter or less flexible (i.e., stiffer)
chains would be expected to have higher affinity for the PSD-95
partner than would a more flexible chain. The results of our
channel C-terminal tails in which most of the intrinsically disor-
dered segment, but not the PDZ-binding motif, was eliminated or
replaced by the globular folded cellulose-binding domain captured
PDZ12to a much greater extent than did the full length ShB-C
protein. In addition, when fused to gliotactin, an intrinsically
disordered chain identical in length yet more flexible than ShB-C,
the Shaker PDZ-binding motif bound less PDZ12than did ShB-C.
In line with these results, systematic shortening of the ShB-C chain
further revealed a monotonic increase in the amount of PDZ12
captured (not shown). Thus, properties associated with polymer
chain chemistry, in particular chain length and conformational
entropy, modulate the interaction strength of the channel C-
terminal tail with the PSD-95 protein. Combined with limited
proteolysis analysis showing that the random chain of ShB-C
acquires no structure upon binding to PDZ12(SI Text), it appears
that ShB-C behaves as an entropic chain, providing the orienta-
scaffold protein partner.
As such, our results support the fishing rod model for Kv
this model is analogous to the ‘‘ball-and-chain’’ model for channel
inactivation (31). In the latter, an intrinsically disordered chain,
harboring at its end the ‘‘ball’’ peptide sequence, serves as an
an intramolecular binding reaction between the chain-linked ball
In the proposed intermolecular fishing rod mechanism, the length
of the intrinsically disordered C-terminal entropic chain of the
with the PSD-95 scaffold protein. (A) SDS/PAGE analysis of eluted fractions of
protein (see Materials and Methods). Lanes 1–5 correspond to pulldown exper-
iments using (i) beads alone or beads containing the following chains as bait: (ii)
ShB-C, (iii) the ShB-C protein mutated in its PDZ-binding motif, (iv) an ShB-C
the 11 last amino acids, (v) an ShB-C mutant protein in which the intrinsically
disordered segment but not the PDZ-binding motif was replaced by the folded
intrinsically disordered segment but not the PDZ-binding motif was replaced by
the intrinsically disordered C-terminal domain of gliotactin (Gli-C). (B) Densito-
metric analysis of the results presented in A. Each reported value represents an
average of eight independent measurements. Differences in the amount of
captured PDZ12in each category, relative to ShB-C, were all found to be statisti-
cally significant, as judged by two-sided Student’s t test. Because multiple com-
parisons of ShB-C to the other protein chains are involved, we followed Bonfer-
roni’s correction and used a more stringent criteria to reject the null hypothesis
that the two compared groups are identical, based on a P value ?1%.
The intrinsically disordered nature of ShB-C modulates its interaction
www.pnas.org?cgi?doi?10.1073?pnas.0704059104Magidovich et al.
channel was set during evolution so as to achieve a relatively short Download full-text
interaction time between both proteins, giving rise to affinity in the
moderate (micromolar) to low (millimolar) range (E.M., unpub-
lished results) (24, 30). Such affinities are typical for dynamic
processes, such as postsynaptic signaling and plasticity, where
transient binding and unbinding reactions play an important role in
the system dynamics (33).
Finally, one can ask whether insight into the mode of interaction
of other ion channels with their cognate PDZ domain-containing
scaffold proteins can be inferred from our bioinformatics and
experimental results addressing the Kv channel–PSD-95 interac-
tion. We find, using sequence analysis, that other PDZ-binding
motif-containing ion channels previously reported to interact with
the PDZ domains of scaffold proteins (34–39), including voltage-
gated sodium channels, NMDA receptors, glutamate receptor
subunits, transient receptor potential channels, inward-rectifier
potassium channels, and aquaporins, are all predicted to bear
intrinsically disordered, unfolded C-terminal domains (not shown).
Accordingly, we propose intrinsically disordered protein segments
to play a general role in mediating channel clustering by scaffold
The results presented in the current study provide direct func-
tional evidence for the involvement of intrinsically disordered
protein segments in processes related to synapse assembly, main-
a protein involved in synapse formation in the brain and implicated
in Alzheimer’s disease (40), and the C-terminal segment of glio-
tactin (23), a protein belonging to the neural cell adhesion protein
family involved in the formation of glutamatergic synapses during
development, have been reported to be intrinsically disordered. In
neither case, however, was a link between the intrinsically disor-
dered character of the protein and its role in synapse formation
established. Considering the important roles intrinsically disor-
protein domains in neuronal systems merits further investigation.
Materials and Methods
For descriptions of molecular biology, protein purification, analyt-
ical ultracentrifugation, CD and1H-NMR spectroscopies, disorder
prediction, cell culture and transformation, confocal microscopy,
and limited proteolysis procedures, refer to SI Text. Other proce-
dures are described below.
Analytical Size-Exclusion Chromatography. For Stokes radius deter-
mination, gel filtration of ShB-C and standard molecular weight
marker proteins was performed on an analytic size-exclusion
column (TSK gel G3000SW column; Tosohass, Tokyo, Japan),
preequilibrated in buffer A (20 mM imidazole/0.3 M NaCl/50
mM Tris?HCl, pH 8) and run at a flow rate of 0.5 ml/min at room
Pharmacia, Uppsala, Sweden). The elution volumes of the
standard and ShB-C proteins (Ve) were converted into mobility-
factor parameters, Kav, using the following equation: Kav ?
(Ve? Vo)/(Vt? Vo), where Vois the column exclusion volume
(5.3 ml), and Vt is the total column bed volume. The Stokes
radius (RST) of ShB-C was estimated using a linear calibration
plot of RSTvs. (?log Kav)1/2(23), obtained with the standard
globular bovine proteins pancreatic ribonuclease A, chymotryp-
sinogen A, ovalbumin, and serum albumin. The theoretical
Stokes radii of native (RST
determined as described (21).
N) or fully unfolded (RST
RC) proteins were
Pulldown Assays. To qualitatively detect interactions between the
PSD-95 and ShB-C proteins, batch-mode pulldown experiments
were performed. Briefly, Ni2?-NTA beads (100 ?l; Qiagen, Chats-
with His6-tagged ShB-C (50 ?M) for 15 min at room temperature
with gentle rocking. After loading of the ShB-C stationary phase,
ShB-C and then challenged for 5 min with a mobile-phase solution
containing either crude soluble protein extracts of PSD-95-GFP-
protein (both prepared in buffer A). After binding, the beads were
washed five times with buffer A followed by elution using buffer A
containing 500 mM imidazole. Captured proteins were detected by
using standard SDS/PAGE (Fig. 5) or Western blot (Fig. 4B)
analyses. The amounts of PDZ12protein captured by either ShB-C
or mutants thereof were quantified densitometrically.
We thank Dr. H. Shumueli for technical assistance and Dr. U. Isacoff
(University of California, Berkeley) for kindly providing the ShB-C-CD8
clone. This research was supported by Israel Science Foundation Grant
323/04 (to O.Y). O.Y is the incumbent of the Belle and Murray Nathan
Career Development Chair in Neurobiology.
1. Sigworth FJ (1994) Q Rev Biophys 27:1–40.
2. Yellen G (1998) Q Rev Biophys 31:239–295.
3. Bezanilla F (2000) Physiol Rev 80:555–592.
4. Hille B (2001) Ion Channels of Excitable Membranes (Sinauer, Sunderland, MA).
5. Tejedor FJ, Bokhari A, Rogero O, Gorczyca M, Zhang J, Kim E, Sheng M,
Budnik V (1997) J Neurosci 17:152–159.
6. Zito K, Fetter RD, Goodman CS, Isacoff EY (1997) Neuron 19:1007–1016.
7. Kim E, Niethammer M, Rothschild A, Jan YN, Sheng M (1995) Nature
8. Tiffany AM, Manganas LN, Kim E, Hsueh YP, Sheng M, Trimmer JS (2000)
J Cell Biol 148:147–158.
9. Ruiz-Canada C, Koh YH, Budnik V, Tejedor FJ (2002) J Neurochem 82:1490–1501.
10. Magidovich E, Fleishman SJ, Yifrach O (2006) Bioinformatics 22:1546–1550.
11. Uversky VN, Gillespie JR, Fink AL (2000) Proteins 41:415–427.
12. Radivojac P, Iakoucheva LM, Oldfield CJ, Obradovic Z, Uversky VN, Dunker
AK (2007) Biophys J 92:1439–1456.
13. Fink AL (2005) Curr Opin Struct Biol 15:35–41.
14. Romero P, Obradovic Z, Li X, Garner EC, Brown CJ, Dunker AK (2001)
15. Tompa P (2002) Trends Biochem Sci 27:527–533.
16. Uversky VN (2002) Protein Sci 11:739–756.
17. Dyson HJ, Wright PE (2005) Nat Rev Mol Cell Biol 6:197–208.
18. Long SB, Campbell EB, Mackinnon R (2005) Science 309:897–903.
Natl Acad Sci USA 100:12607–12612.
20. Fukuchi S, Homma K, Minezaki Y, Nishikawa K (2006) J Mol Biol 355:845–857.
21. Uversky VN (2002) Eur J Biochem 269:2–12.
22. Scherf T, Kasher R, Balass M, Fridkin M, Fuchs S, Katchalski-Katzir E (2001)
Proc Natl Acad Sci USA 98:6629–6634.
23. Zeev-Ben-Mordehai T, Rydberg EH, Solomon A, Toker L, Auld VJ, Silman I,
Botti S, Sussman JL (2003) Proteins 53:758–767.
24. Long JF, Tochio H, Wang P, Fan JS, Sala C, Niethammer M, Sheng M, Zhang
M (2003) J Mol Biol 327:203–214.
25. Kennedy MB (2000) Science 290:750–754.
26. Baron MK, Boeckers TM, Vaida B, Faham S, Gingery M, Sawaya MR, Salyer
D, Gundelfinger ED, Bowie JU (2006) Science 311:531–535.
27. Garner CC, Nash J, Huganir RL (2000) Trends Cell Biol 10:274–280.
28. Sheng M, Sala C (2001) Annu Rev Neurosci 24:1–29.
29. Kim E, Sheng M (2004) Nat Rev Neurosci 5:771–781.
30. Harris BZ, Lim WA (2001) J Cell Sci 114:3219–3231.
31. Hoshi T, Zagotta WN, Aldrich RW (1990) Science 250:533–538.
32. Zhou M, Morais-Cabral JH, Mann S, MacKinnon R (2001) Nature 411:657–661.
33. Sheng M, Kim MJ (2002) Science 298:776–780.
34. Kornau HC, Schenker LT, Kennedy MB, Seeburg PH (1995) Science 269:1737–
35. Tang Y, Tang J, Chen Z, Trost C, Flockerzi V, Li M, Ramesh V, Zhu MX (2000)
J Biol Chem 275:37559–37564.
36. Leonoudakis D, Mailliard W, Wingerd K, Clegg D, Vandenberg C (2001) J Cell
37. Adams ME, Mueller HA, Froehner SC (2001) J Cell Biol 155:113–122.
38. Gee SH, Madhavan R, Levinson SR, Caldwell JH, Sealock R, Froehner SC
(1998) J Neurosci 18:128–137.
39. Xia J, Zhang X, Staudinger J, Huganir RL (1999) Neuron 22:179–187.
40. Weinreb PH, Zhen W, Poon AW, Conway KA, Lansbury PT, Jr (1996)
41. Uversky VN, Oldfield CJ, Dunker AK (2005) J Mol Recognit 18:343–384.
42. Dyson HJ, Wright PE (2002) Curr Opin Struct Biol 12:54–60.
Magidovich et al.
August 7, 2007 ?
vol. 104 ?
no. 32 ?