The occludin and ZO-1 complex, defined by small angle X-ray scattering and NMR, has implications for modulating tight junction permeability.
ABSTRACT Tight junctions (TJs) are dynamic cellular structures that are critical for compartmentalizing environments within tissues and regulating transport of small molecules, ions, and fluids. Phosphorylation-dependent binding of the transmembrane protein occludin to the structural organizing protein ZO-1 contributes to the regulation of barrier properties; however, the details of their interaction are controversial. Using small angle X-ray scattering (SAXS), NMR chemical shift perturbation, cross-saturation, in vitro binding, and site-directed mutagenesis experiments. we define the interface between the ZO-1 PDZ3-SH3-U5-GuK (PSG) and occludin coiled-coil (CC) domains. The interface is comprised of basic residues in PSG and an acidic region in CC. Complex formation is blocked by a peptide (REESEEYM) that corresponds to CC residues 468-475 and includes a previously uncharacterized phosphosite, with the phosphorylated version having a larger effect. Furthermore, mutation of E470 and E472 reduces cell border localization of occludin. Together, these results localize the interaction to an acidic region in CC and a predominantly basic helix V within the ZO-1 GuK domain. This model has important implications for the phosphorylation-dependent regulation of the occludin:ZO-1 complex.
- SourceAvailable from: Fariba Rezaee[Show abstract] [Hide abstract]
ABSTRACT: Epithelial permeability is a hallmark of mucosal inflammation, but the molecular mechanisms involved remain poorly understood. A key component of the epithelial barrier is the apical junctional complex that forms between neighboring cells. Apical junctional complexes are made of tight junctions and adherens junctions and link to the cellular cytoskeleton via numerous adaptor proteins. Although the existence of tight and adherens junctions between epithelial cells has long been recognized, in recent years there have been significant advances in our understanding of the molecular regulation of junctional complex assembly and disassembly. Here we review current thinking about the structure and function of the apical junctional complex in airway epithelial cells, emphasizing translational aspects of relevance to cystic fibrosis and asthma. Most work to-date has been conducted using cell culture models, but technical advancements in imaging techniques suggest that we are on the verge of important new breakthroughs in this area in physiological models of airway diseases.American Journal of Respiratory Cell and Molecular Biology 01/2014; · 4.15 Impact Factor
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ABSTRACT: Retinal ischemia-reperfusion (IR) induces neurodegenaration as well as blood-retinal barrier (BRB) breakdown causing vascular permeability. Whereas the neuronal death has been extensively studied, the molecular mechanisms related to BRB breakdown in IR injury remain poorly understood. In this study, we investigated the early changes in tight junctional (TJ) proteins in response to IR injury. Ischemia-reperfusion injury was induced in male rat retinas by increasing the intraocular pressure for 45 minutes followed by natural reperfusion. The results demonstrate that IR injury induced occludin Ser490 phosphorylation and ubiquitination within 15 minutes of reperfusion with subsequent vascular permeability. Immunohistochemical analysis revealed a rapid increase in occludin Ser490 phosphorylation and loss of Zonula occludens-1 (ZO-1) protein, particularly in arterioles. Ischemia-reperfusion injury also rapidly induced the activation and phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) at tyrosine 1175. Blocking vascular endothelial growth factor (VEGF) function by intravitreal injection of bevacizumab prevented VEGFR-2 activation, occludin phosphorylation, and vascular permeability. These studies suggest a novel mechanism of occludin Ser490 phosphorylation and ubiquitination downstream of VEGFR2 activation associated with early IR-induced vascular permeability.Journal of Cerebral Blood Flow & Metabolism advance online publication, 8 January 2014; doi:10.1038/jcbfm.2013.230.Journal of cerebral blood flow and metabolism: official journal of the International Society of Cerebral Blood Flow and Metabolism 01/2014; · 5.46 Impact Factor
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ABSTRACT: To investigate the protective effects of combinations of probiotic (Bifico) on interleukin (IL)-10-gene-deficient (IL-10 KO) mice and Caco-2 cell monolayers. IL-10 KO mice were used to assess the benefits of Bifico in vivo. IL-10 KO and control mice received approximately 1.5 × 10(8) cfu/d of Bifico for 4 wk. Colons were then removed and analyzed for epithelial barrier function by Ussing Chamber, while an ELISA was used to evaluate proinflammatory cytokines. The colon epithelial cell line, Caco-2, was used to test the benefit of Bifico in vitro. Enteroinvasive Escherichia coli (EIEC) and the probiotic mixture Bifico, or single probiotic strains, were applied to cultured Caco-2 monolayers. Barrier function was determined by measuring transepithelial electrical resistance and tight junction protein expression. Treatment of IL-10 KO mice with Bifico partially restored body weight, colon length, and epithelial barrier integrity to wild-type levels. In addition, IL-10 KO mice receiving Bifico treatment had reduced mucosal secretion of tumor necrosis factor-α and interferon-γ, and attenuated colonic disease. Moreover, treatment of Caco-2 monolayers with Bifico or single-strain probiotics in vitro inhibited EIEC invasion and reduced the secretion of proinflammatory cytokines. Bifico reduced colon inflammation in IL-10 KO mice, and promoted and improved epithelial-barrier function, enhanced resistance to EIEC invasion, and decreased proinflammatory cytokine secretion.World Journal of Gastroenterology 04/2014; 20(16):4636-4647. · 2.55 Impact Factor
The occludin and ZO-1 complex, defined by small
angle X-ray scattering and NMR, has implications
for modulating tight junction permeability
Brian R. Tasha,1,2, Maria C. Bewleya,1, Mariano Russob, Jason M. Keilc, Kathleen A. Griffina, Jeffrey M. Sundstromd,
David A. Antonettic, Fang Tiana, and John M. Flanagana,3
aDepartments of Biochemistry and Molecular Biology,
Medicine, H171, 500 University Drive, Hershey, PA 17033; and
Ann Arbor, MI 48105
bCellular and Molecular Physiology, and
cDepartment of Ophthalmology and Visual Sciences, Kellogg Eye Center, 1000 Wall Street,
dOphthalmology, Pennsylvania State University College of
Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved May 21, 2012 (received for review January 2, 2012)
Tight junctions (TJs) are dynamic cellular structures that are critical
for compartmentalizing environments within tissues and regulat-
ing transport of small molecules, ions, and fluids. Phosphoryla-
tion-dependent binding of the transmembrane protein occludin
to the structural organizing protein ZO-1 contributes to the regula-
tion of barrier properties; however, the details of their interaction
are controversial. Using small angle X-ray scattering (SAXS), NMR
chemical shift perturbation, cross-saturation, in vitro binding, and
site-directed mutagenesis experiments. we define the interface
between the ZO-1 PDZ3-SH3-U5-GuK (PSG) and occludin coiled-coil
(CC) domains. The interface is comprised of basic residues in PSG
and an acidic region in CC. Complex formation is blocked by a pep-
tide (REESEEYM) that corresponds to CC residues 468–475 and
includes a previously uncharacterized phosphosite, with the phos-
phorylated version having a larger effect. Furthermore, mutation
of E470 and E472 reduces cell border localization of occludin. To-
gether, these results localize the interaction to an acidic region
in CC and a predominantly basic helix V within the ZO-1 GuK
domain. This model has important implications for the phosphor-
ylation-dependent regulation of the occludin∶ZO-1 complex.
membrane-associated guanylate kinase ∣ tricellulin ∣ calmodulin
lial or epithelial cells (1, 2). These barriers function in a range of
tissues, including the vasculature of the central nervous system,
kidney, and gut epithelium. Dysregulation of barrier properties is
associated with a host of disease states including cancer, stroke,
diabetic retinopathy, and inflammatory bowel syndrome (3–5).
Furthermore, viral and bacterial pathogens exploit specific TJ
proteins to gain access to host cells. Thus, understanding the con-
tribution of TJ components in barrier formation and regulation
may aid the development of therapies to restore barrier prop-
erties, control barrier properties to promote drug delivery to re-
gions of the CNS, and for the development of novel antibacterial
and antiviral compounds.
Occludin is an integral membrane, vesicle-trafficking, MAL
and related proteins for vesicle trafficking and membrane link
(MARVEL) protein (6), that has a role in regulating TJ proper-
ties (7). For example, in endothelial cells, it regulates TJ barriers
in response to cytokines, such as IFN-γ (8, 9), and growth factors,
such as VEGF (10). Removal of the cytoplasmic C-terminal
coiled coil domain (CC; residues 413–522) of occludin leads to
cytoplasmic localization with increased tracer flux through the
junctions and an inability to maintain the apical localization of
marker proteins (11, 12), identifying the CC as a key element
in regulation (13). The CC directly associates with the SH3-U5-
GuK domains of the membrane-associated guanylate kinase
homolog (MAGUK) protein, ZO-1 (14). ZO proteins are
characterized by their core PDZ3-SH3-GuK (PSG) domains,
ight junctions (TJs) are highly polarized gates that control the
flux of fluids, proteins, and even ions across sheets of endothe-
preceded by two additional PDZ domains and separated by
unique regions (U1–6), each with distinct functions (15).
The interaction of ZO-1 and occludin is modulated by phos-
phorylation of residues in CC that affects the properties of TJs
(7). Biochemical and biophysical experiments have led to two dis-
tinct models for the complex (Fig. 1). In the first, (Model 1) a
cluster of basic residues on CC (K433, K444, K485, K488, K504,
and K511) form the interface with ZO-1 (16). In the second
(Model 2), the interface was formed between the acidic surface
of CC and regions of the ZO-1 U5 and GuK domain (17–19). The
crystal structures of ZO-1 PSG and SH3-GuK domains (20–22)
seemed to support the Model 2, since the surface of the GuK
domain is largely basic (Fig. 1B). The potential contributions of
the ZO-1 U6 (not part of the minimal CC binding domain) or U5
motifs to the interface are unknown as they are absent from the
structures. Thus, no clear consensus exists on how occludin and
ZO-1 interact or how phosphorylation modulates this interaction.
In this study, we present a structural model for the minimal
complex between occludin and ZO-1 (CC∶PSG) where residues
468–475 (REESEEYM) in the CC acidic head directly interact
with helix V of ZO-1 GuK. This model is consistent with our
data from SAXS, NMR, in vitro, and ex vivo binding studies
and in transiently transfected Madin–Darby canine kidney
(MDCK) cells. Furthermore, we found that a phosphopeptide
(YREEpS471EEYM) bound approximately 20-fold tighter than
its nonphosphorylated equivalent to PSG, implicating S471 phos-
phorylation in regulating binding. In light of these results, we
discuss roles for CC phosphorylation in complex stability and
CC and PSG Form an Extended Complex in Solution. To discriminate
between the opposing models for the ZO-1∶occludin complex
(16, 19), we measured the SAXS curves of the minimal binding
domains (CC and PSG) alone and in their binary complex
(Fig. 2A). From the Guinier plot, all samples were monodisperse
(Fig. 2A and SI Appendix, Fig. S1A) and monomeric, based on
the relative scattering intensities at zero angle and Porod volumes
Authorcontributions:B.R.T.,M.C.B.,J.M.S., D.A.A.,F.T.,and J.M.F. designedresearch; B.R.T.,
M.C.B., M.R., J.M.K., K.A.G., F.T., and J.M.F. performed research; B.R.T., M.C.B., K.A.G.,
J.M.S., D.A.A., F.T., and J.M.F. contributed new reagents/analytic tools; B.R.T., M.C.B.,
M.R., J.M.K., K.A.G., J.M.S., D.A.A., F.T., and J.M.F. analyzed data; and B.R.T., M.C.B.,
D.A.A., F.T., and J.M.F. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1B.R.T. and M.C.B. contributed equally to this work.
2Present address: Renal Electrolyte and Hypertension Division, University of Pennsylvania,
Philadelphia, PA 19104.
3To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/
July 3, 2012
of CC and PSG. The molecular weight of the CC∶PSG complex
was consistent with a 1∶1 stoichiometry, and this was confirmed
in a titration experiment (SI Appendix, Fig. S1B). The radius of
gyration (Rg) extrapolated to zero concentration of CC and
PSG were 23.2 ? 0.4 Å (approximately 24 Å, 1WPA.PDB,
3G7C.PDB) (7, 16) and 28.9 ? 0.3 Å (approximately 29 Å,
3SHW.PDB) (22), respectively, and 35.6 ? 0.6 Å for the complex
(Fig. 2A). These values correspond to a centers-of-mass separa-
tion of 51 ? 2 Å from the parallel axis theorem (23), suggesting
an extended complex. Further, the distance distribution func-
tions, PðrÞ, and maximum chord length, Dmax, for the CC and
PSG alone and in complex support this conclusion (Fig. 2B).
The shape of the PðrÞ curve for CC is indicative of a rod
(Dmax¼ 80 Å), while PSG is more compact (Dmax¼ 95 Å),
consistent with their crystal structures (CC Dmaxapproximately
75 Å, 1XAW.PDB; PSG Dmaxapproximately 93 Å, 3SHW.PDB).
The complex is significantly longer (Dmax¼ 140 Å).
The SAXS-derived molecular envelope of CC was rod-like
(Fig. 2C), while PSG was boot-shaped in high or low NaCl
(Fig. 2D and SI Appendix, Fig. S1 C and D) with the SH3 at
the heel, GuK forming the ankle, and PDZ3 the toes. The envel-
ope of the complex was extended at the ankle, presumably due to
binding of the CC (Fig. 2E). Crystal structures of CC and PSG
were fit as rigid molecules to the SAXS data (SI Appendix,
Fig. S2) (24), either unconstrained or constraining the relative
placement of CC and PSG (SI Appendix, Fig. S2). Constraining
either E470 or 472 (CC, acidic head) to be <7 Å from K760
(PSG, GuK, helix V) resulted in the best fit to the data (SI
Appendix, Fig. S2) and gave marginally better χ2values than with-
out constraints. This arrangement is consistent Model 2 (17–19).
Constraints involving K433 (CC), to test Model 1, gave much
poorer fits to the scattering data (SI Appendix, Fig. S2). More-
over, constraining the model to be a homodimer of PSG, rather
than a CC∶PSG complex, fit the data poorly (χ2¼ 3.1), consis-
tent with the observation that dimerization occurs through PDZ2,
which was not in our construct (25).
The binding of PSG to CC was characterized in a quantitative
capture assay (SI Appendix, Fig. S3). PSG (2 μM) was soluble and
did not bind to GST beads alone or in the presence of GST, even
in 50 mM NaCl. It was efficiently captured on these beads by
GST-CC (SI Appendix, Fig. S3A, 2 μM). The amount captured
was saturable and the maximum relative intensity for the cap-
tured PSG and CC was consistent with a 1∶1 stoichiometry at
50 mM NaCl (SI Appendix, Fig. S3 B and C). Under these con-
ditions, the apparent dissociation constant, Kd, was 1.7 ? 0.2 μM
and increased with NaCl concentrations (SI Appendix, Fig. S3C,
6 ? 1 μM at 100 mM NaCl; 12 ? 2 μM at 150 mM NaCl,). These
data indicate a net displacement, Δn, of1.7 Naþplus Cl−in form-
ing the GST-CC∶PSG complex (SI Appendix, Fig. S3C), consis-
tent with an ionic interface.
CC Binds to Basic Residues in Helix V of the ZO-1 GuK Domain. To
define the CC binding surface on PSG, we focused on the region
in and around residues 749–768 (helix V, GuK), a conserved, pre-
dominantly basic region that contributes to calmodulin (CaM)
Residues characterized in detail are labeled and shown in stick representa-
tion. (A, Top) Ribbon diagram of CC. K433, K444, K485, K488, K504, and
K511 (basic face) (16) are blue. (Middle) Surface representation with arrows
and a circle highlighting the basic (blue) and acidic (red) surfaces, respec-
tively. (Bottom) Residues in the loop between the first two helices are drawn
as sticks (acidic, red; hydrophobic, yellow; phosphorylatable Ser, purple). All
other residues are shown as a cyan ribbon. (B, Top) Ribbon diagram of PSG
(PDZ3, orange; SH3, mid-blue; GuK, green) showing basic residues in helix V
(blue). The asterisk (*) denotes the ends of the U5 motif. (Middle) Surface
representation colored as in A. A circle highlights the region around helix
V. (Bottom) Residues within helix V involved in CaM binding (20).
Ribbon diagrams showing location of key residues in CC and PSG.
(A) Plot of log of scattered intensity versus scattering angle, q, for CC (black),
PSG (red), and their binary complex (blue); (Inset) Guinier plot for each. (B) PðrÞ
curves. (C) Fit of CC (cyan ribbon), (D) PSG (PDZ3, red; SH3, blue; GuK, green),
and (E) the binarycomplex, into their respectiveabinitioenvelopes (beige net).
Analysis of the SAXS data for CC, PSG, and their binary complex.
dition of increasing amounts of purified CaM inhibits the capture of PSG by
GST-CC. A representative Coomassie-stained gel of the GST-CC and PSG
bound to glutathione resin as a function of added CaM with a summary
of the amount of captured PSG relative to GST-CC relative (n ¼ 3). For statis-
tical significance: * p < 0.05, ** p < 0.01, and *** p < 0.001. (B) The charge
swaps in helix V of PSG 2D/E and 3D/E cannot bind CaM (20) and are not
captured by GST-CC on glutathione beads.
CaM competes with CC for binding to the ZO-1 GuK domain. (A) Ad-
www.pnas.org/cgi/doi/10.1073/pnas.1121390109 Tash et al.
binding (20). The CaM∶PSG complex by SAXS was elongated
(Rg35.4 ? 0.4 Å) with a 1∶1 stoichiometry. The SAXS-derived
envelope suggested that CaM bound to PSG in a similar position
to CC (SI Appendix, Fig. S4). Further, CaM inhibited, in a dose-
dependent manner, the capture of PSG by GST-CC (Fig. 3A),
suggesting that they share a common binding site on ZO-1.
The basic residues in helix V GuK are a primary determinant
of CaM binding (20). Therefore, we tested the effect on CC bind-
ing of the variants used in the CaM studies [(20); K749D/R752D/
K753E (PSG 3D/E) and K760E/K763E (PSG 2D/E) in helix Vof
ZO-1 GuK)]. Both PSG variants were captured in significantly
reduced amounts by GST-CC (Fig. 3B), demonstrating that
CC and CaM are direct competitors for PSG and share a binding
site that includes basic residues within helix V GuK.
The Acidic Head of CC Forms the Interface. In the optimal SAXS-
based model, the acidic head of CC forms part of the ZO-1 bind-
ing site; however, the data does not conclusively exclude binding
via the opposite end of CC near its N and C termini (residues
414–439 and 508–522). To resolve this question, we performed
NMR chemical shift perturbation and cross-saturation experi-
ments. Backbone resonance assignment of 109 of 112 residues
in CC was accomplished with the transverse relaxation optimized
spectroscopy (TROSY) form of standard triple resonance experi-
ments (SI Appendix, Fig. S5) (26, 27) and provided the basis
for identification of the CC-binding surface. Chemical shift per-
turbation experiments comparing CC and CC∶PSG identified
two clusters of residues that showed small but detectable changes
in the TROSY spectrum. The first cluster was located within helix
1 (N454, E456, and R459) and the second in the acidic head
(Fig. 4A and SI Appendix, Fig. S6; L464, D465, Y467, E470,
S471, E473, M475, and A478). No clusters in chemical shift per-
turbations were observed for residues at the opposite end of the
CC. The observed chemical shifts are relatively small because the
CC∶PSG interaction is most likely dominated by side chains with
little perturbation of the backbone environment. There was
further evidence of complex formation when comparing the
TROSY spectra of15N,2H labeled (random fractionally deuter-
ated) PSG with and without unlabeled CC (SI Appendix, Fig. S7).
Upon addition of CC numerous resonances appeared between
8.1–8.5 ppm1H and 120–126 ppm15N and a large peak asso-
ciated with an unfolded resonance signal disappeared, suggesting
some structural rearrangement in PSG upon complex formation.
To determine whether the chemical shift perturbations result
from direct binding or allosteric effects, NMR cross-saturation
experiments (28) were performed. Perdeuterated ½15N?-CC was
bound to protonated, unlabeled PSG at 2∶1, 1∶1, and 1∶2 molar
ratios. Due to the tendency of free PSG to aggregate at high pro-
tein concentrations and prolonged incubation at 27°C, total PSG
was kept at 100 μM and short collection times were used (<24 h).
To overcome the sensitivity limitations, buffers containing 40%
and 70% D2O were used instead of the more usual 90% D2O.
To avoid potential false positives arising from the spin diffusion
effect, we used a conservative criterion for identifying perturbed
with ZO-1. (A) Close-up of TROSY spectrum of per-
deuterated ½15N?-apo (red) CC overlaid with PSG-
bound perdeuterated ½15N?-CC (black) showing the
shifts for residues affected by the addition of CC
(Y467, A478) and those unaffected (K433). (B) 1D
traces of the residues in A from cross-saturation re-
laxation experiments for saturation at 15 ppm (red)
and 1 ppm (black). (C) The in vitro capture efficiency
of PSG by GST-CC is reduced by charge reversals at
residues 465∕469, 470, 472, and 473 in the acidic
head of CC. (D) The variant GST-CC, except K433D,
had a similar effect upon the capture of full-length
ZO-1 from MDCK lysates. Relative binding (below
the gel) was the intensity ratio of the ZO-1 band
in each lane and the wt condition.
The acidic head of CC mediates interaction
Tash et al. PNAS
July 3, 2012
resonances. L464, D466, Y467, and A478 were at the interface in
these experiments (Fig. 4B and SI Appendix, Fig. S8). No residues
around the N and C termini showed significant cross-saturation
relaxation effects. Taken together, the SAXS and NMR experi-
ments demonstrate that CC binds with its acidic head in proximity
to PSG, while the opposite end, containing its N and C termini, is
furthest from this interface.
To probe the contribution of specific residues in the acidic
head tocomplex stability, the effect ofsingle (E470K,E472K, and
E473K) and double (D465K/E469K) substituted variants of GST–
CC were evaluated in PSG capture assays (Fig. 4C). The E473K
variant marginally reduced capture (82% of wt, p < 0.001) while
more dramatic reductions were observed for the E470K, E472K,
and D465K/E469K variants [43%, 47%, and 23% of wt (all
p < 0.001), respectively]. The E470K, E472K, and D465K/E469K
GST-CC variants also showed reduced capture of full-length ZO-1
from MDCK cell lysates (Fig. 4D; 18%, 49%, and 20% of wt, re-
spectively, p < 0.001) confirming the importance of the negative
charges on CC in mediating its interaction with ZO-1. By contrast,
the K433D substitution, located on the basic face of CC, displayed
opposite effects in vitro (Fig. 4C) and in lysate (Fig. 4D). In vitro,
K433D enhanced capture of PSG (136% of wt, p < 0.001) but, as
reported previously (16), it abolished the interaction with full-
length ZO-1 (14% of wt, p < 0.001), suggesting that K433 may
be involved in a contact present in full-length ZO-1 (29) but not
in PSG. A possible candidate is the acidic U6 motif (residues 803–
888), that regulates complex stability but is not required for the
binding oftheminimalinteractingdomains (29).However,wecan-
not exclude other possibilities, such as post-translational modifica-
tions of ZO-1 or additional factors present in the extract.
E470 and E472 in CC Contribute to TJ Localization in Cells. Since
E470K and E472K substitutions had profound effects on ZO-1
binding, full-length V5-tagged occludin or variants containing
these substitutions were transiently transfected into MDCK cells.
Expression of wt occludin resulted in immunostaining at the cell
membrane, with the majority occurring at cell∶cell contacts,
colocalizing with ZO-1 and endogenous occludin (Fig. 5A). The
3D rendering of these confocal images revealed good colocaliza-
tion of wt occludin with ZO-1 at the apical membrane border
(Fig. 5B). By contrast, the E470K and E470K/E472K occludin
variants showed increased localization in the cytoplasm and at the
lateral border (Fig. 5B) and reduced staining at cell contacts, with
loss of ZO-1 colocalization. To quantify the change in localiza-
tion, the ratio of Triton X-100-soluble and insoluble expressed
occludin was compared to ZO-1. As expected, the majority of wt
occludin was found in the Triton X-100-insoluble fraction, along
with ZO-1. However, the E470K and E470K/E472K variants were
located in the soluble fraction, with a 66% (p < 0.01) decrease
inthe ratioofTritonX-100-insoluble∶soluble occludinfor thedou-
ble variant (SI Appendix, Fig. S9). These results suggest that the
interaction of ZO-1 with the acidic head of CC is necessary for
either the transport to, or maintenance of, occludin at TJs.
Phosphorylation of S471 May Enhance Occludin∶ZO-1 Complex Stabi-
lity. The two acidic residues, E470 and E472, with a key role in
localizing occludin at TJs, flank S471, a previously identified
phosphosite (7). To investigate the effect of S471 phosphoryla-
tion, occludin containing a S471D substitution (S471D) was tran-
siently transfected into MDCK cells. Using confocal microscopy,
S471D demonstrated good apical border colocalization with
ZO-1 (Fig. 5).
To further define the effect of S471 phosphorylation, peptides
with and without a phosphogroup on S471 (residues 468–475,
REESEEYM, and REEpSEEYM) were tested for their ability
to inhibit PSG capture by GST-CC (Fig. 6A). Both peptides
significantly inhibited the capture of PSG [19% and 33% of wt,
(p < 0.001), respectively], with the phosphorylated peptide being
more efficient than the nonphosphorylated peptide (p < 0.01).
This observation was similar in experiments to capture endogen-
ous ZO-1 from MDCK cell lysates (Fig. 6B; 32% and 41%,
respectively, p < 0.05). By contrast, peptides containing S490
head disrupt occludin localization. Confocal images
of immunocytochemistry for ZO-1, occludin, and
MDCK cells transiently transfected with V5 tagged
wt, E470K, E470K/E472K, or S471D variants of occlu-
din. (A) Max projected confocal image. Arrows indi-
cate failure of E470K/E472K mutant to colocalize
with ZO-1. There is good localization for S471D.
Bar represents 10 μm. (B) Topical surface of V5 stain-
ing and V5 staining plus ZO-1 rendered in 3D
volume. Bar represents 2 μm.
Charge reversal mutations in the acidic
www.pnas.org/cgi/doi/10.1073/pnas.1121390109 Tash et al.
(residues 486–494, QVKGSADYK and QVKGpSADYK), a sec-
ond validated phosphosite in CC (7, 10) and a control peptide
encompassing K433 (residues 430–438, QLYKRNFDT) did not
significantly alter capture efficiency in vitro or ex vivo.
To quantitate binding of the S471-containing peptides, a fluor-
escently labeled peptide corresponding to residues 467–475 CC
[FITC—(miniPEG)—YREESEEYM] was synthesized. Based
upon the changes in fluorescent anisotropy of this peptide as a
function of PSG concentration, the Kdfor labeled peptide bind-
ing was 1.8 ? 0.4 μM (Fig. 6C and SI Appendix), although the
fluorophore and the linker appeared to contribute slightly to the
interaction. In competition assays, the equivalent unlabeled pep-
tide (Ac—YREESEEYM) bound with a Kdof 15 ? 3 μM, while
the serine phosphorylated peptide (Ac—YREEpSEEYM) in-
creased the affinity by approximately 20-fold (Kd¼ 0.70?
0.05 μM). By contrast, a scrambled peptide (Ac-EEYRSYMEE)
did not affect binding of the labeled peptide (Fig. 6C). Taken as a
whole, these results confirm that the acidic head of CC directly
mediates its interaction with the GuK domain of PSG with high
affinity and phosphorylation of S471 enhances occludin∶ZO-1
Previous studies of TJs have established a close association
between the scaffolding protein, ZO-1, and the transmembrane
protein, occludin, and identified multiple phosphorylation sites as
potential regulators of their interaction. However, the molecular
details ofthisinteraction were unclear. The resultsdescribed here
suggest a model for complex formation utilizing two distinct in-
terfaces that contain phosphorylatable residues within or around
their periphery (Fig. 7). The core of the interface is electrostatic,
involving D465, E469, E470, E472, and E473 in CC and residues
K749, R752, K753, K760, and K763 in PSG. Consistent with this
model, the stability of the complex is salt-dependent. In addition,
helix V (residues 751–765) (20) is used by two other MAGUK
family members for binding their phosphosites (30, 31). Finally,
the coiled-coil domain of tricellulin, a homolog of occludin pre-
sent at tricellular junctions, lacks the acidic residues in its head
domain and does not interact strongly with PSG in SAXS and
capture assays (SI Appendix, Fig. S10) (32).
The argument for a second binding interaction between occlu-
din and ZO-1 is based upon differences in the effects of some
amino acid substitutions in CC on their capture efficiency of
PSG and longer ZO-1 constructs. Charge reversal substitutions
of lysine residues in the basic face of CC did not affect capture
of PSG in vitro but, consistent with earlier data, showed reduced
capture of longer ZO-1 constructs (1–888 and full-length ZO-1)
that contain U6 (16). Therefore, we speculate there is a second
ionic interface that involves interactions between the basic face of
CC, including K433, and acidic residues on the surface of the full-
length ZO-1, perhaps within the U5 or U6 motifs (16, 29, 33) or
involving a bridge with another cellular factor. The presence of
this secondary interface may enhance the stability of the complex
in the cellular environment and/or contribute to the regulation
of the complex. One very attractive possibility is that binding of
ZO-1 binding to the secondary interface controls access to the
lysine-rich face of CC, which contains seven of the 12 conserved
lysine residues in occludin, and thus its ubiquitin-dependent en-
docytosis observed in endothelial cells (10).
In vivo, the occludin∶ZO-1 interaction is modulated by phos-
phorylation events (7, 34–38). In occludin, four of these sites seg-
regate structurally and possibly functionally—two adjacent to the
primary interaction site (S471 and Y474) in the acidic head and
two in the secondary interface (S490 and S508) on the basic face.
Y474 phosphorylation is associated with binding p85 of phospha-
tidyl inositol 3-kinase at the leading edge of migrating cells (39)
while S490 and S508 are associated with vascular permeability
after VEGF treatment (7) or HIV encephalitis (40). Our results
suggest a role for S471 phosphorylation in ZO-1 binding. The CC
peptide 468–475 containing S471 and Y474, is more effective at
blocking PSG and ZO-1 capture when phosphorylated at S471
(pS471), suggesting that phosphorylation here may enhance com-
plex formation in vivo. Indeed, in MDCK cells, the S471D-occlu-
din variant exhibited at least as good colocalization with ZO-1 as
wt (Fig. 5). The results are consistent with the recent finding
that the GuK domain has evolved as a phosphopeptide-binding
module (31). The Kdfor pS471 peptide binding to PSG (0.7 μM)
is very similar to that of p-LGN binding to SAP97 SH3-GuK
(0.22 μM). However, the Kdvalues of the equivalent nonpho-
sphorylated peptides are different (S471 to PSG, approximately
15 μM; LGN to SPA97, >100 μM). Interestingly, occludin pos-
sesses the requisite arginine at −3 relative to the phosphosite
of GuK binding peptides (31); however, ZO-1 does not contain
the sequence required for high affinity binding. These results are
consistent with S471 phosphorylation strengthening or modulat-
ing binding to ZO-1 rather than providing an all or none binding
motif. By contrast, phosphorylation of S490 reduces its inter-
action with endogenous ZO-1 (7). While the effect of S508 phos-
phorylation on ZO-1 binding is not known, its location adjacent
to K504 and K511, which are required for complex formation
with endogenous ZO-1 (16), suggests that S508 phosphorylation
would reduce ZO-1 binding. Further, phosphorylation of either
PSG. (A) In vitro, only inclusion of peptides (200 μM) spanning residues
the acidic head, 468–475 (?pS471) inhibited capture of PSG by GST-CC on
glutathione beads. (B) In MDCK lysates, only the peptides in A significantly
reduced capture of full-length ZO-1 by GST-CC (n ¼ 3). (C) Equilibrium bind-
ing of the fluorescent-labeled peptide, FITC-(miniPEG)-YREESEEYM, to PSG
in the absence and presence of unlabeled peptides. In all experiments, 3 nM-
labeled peptide was titrated with purified PSG. A representative titration
and fit to a single binding site model is shown (▪, solid line). Inclusion of
a nonfluorescently labeled version of this peptide (○, long dash, weak com-
petition) or this peptide with S471 phosphorylated (▾, short dash, strong
competition) but not a scrambled version of this sequence (Δ, no competi-
tion) reduced PSG binding compared to the unlabeled peptide alone.
A phosphopeptide containing S471 competes with CC binding to
phosphosites on CC. Domains shown as rectangles, colored as Fig. 1. Phospho-
sites are ovals that are shaded when phosphorylated. U5 and U6 are repre-
sented as lines.
Working model of the CC∶PSG interaction and the role of the various
Tash et al. PNAS
July 3, 2012
S490 or S508 correlates with opening of the barrier in endothelial
cells (7, 40), suggesting that the basic face of CC is involved in
regulating TJ barrier properties in vivo. Five additional phospho-
sites that lie in a conserved region just upstream of CC (Y398,
Y402, T403, T404, and T408) also regulate phosphorylation-de-
pendent complex formation. Based on geometric considerations,
they cannot exert their effect directly at the primary interaction
site and may modulate binding through a different site, perhaps
close to the proposed secondary interaction site.
In summary, our model for the complex between CC and PSG,
provides a framework for understanding the functional role of
the occludin∶ZO-1 complex in TJ permeability via occludin phos-
phorylation and offers new insight into the regulation of barrier
properties. These studies highlight the direct interaction of the
CC, through its acidic head, with the basic region surrounding
helix V in PSG. Undoubtedly, additional contacts exist in the
larger TJ complex that contribute to complex organization;
however, the relative contribution and mechanism of action of
occludin phosphorylation on its interaction with ZO-1 is emer-
ging. A complete understanding of the interface may uncover
new therapeutic strategies to selectively alter TJ permeability
in the treatment of disease or delivery of drugs to the central
E. coli expressed human CC and human PSG were expressed as tobacco etch
virus (TEV) protease cleavable His6-tagged fusion proteins; the TEV-cleaved
form of each was used for SAXS and NMR experiments. A GST-tagged version
of the CC (413–522) was expressed and purified using standard protocols (SI
Appendix, Methods) and used in vitro and in cell lysate capture assays. The
final concentrations of proteins used in individual experiments are given in
the text and the figure legends. SAXS experiments were conducted at X9,
National Synchrotron Light Source, Upton, NY, using a standard configura-
tion and software. NMR experiments were conducted on Bruker Avance
600 MHz and 850 MHz spectrometers, equipped with cold probes using stan-
dard protocols. In-cell based assays were conducted with MDCK cells using
standard protocols. Additional details are available in the SI Appendix.
ACKNOWLEDGMENTS. We thank Drs. Marc Allaire and Lin Yang for discussions
and technical assistance with small angle X-ray scattering. We thank the
Pennsylvania Lions Sight Conservation and Eye Research Foundation
(J.M.F.), the American Diabetes Association Grant 7-07-RA-34 (to J.M.F.),
National Eye Institute Grant EY012021 (to D.A.), and the Pennsylvania Tobac-
co Funds (J.M.F., F.T., and D.A.) for financial support. The Kellog Eye Center
imagine core is supported by National Institutes of Health Grants EY07003
and P60DK020572. The National Synchrotron Light Source and beamline
X9 is supported by the US Department of Energy, Office of Science, Office
of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886.
1. Van Itallie CM, Anderson JM (2004) The molecular physiology of tight junction pores.
Physiology (Bethesda) 19:331–338.
2. Forster C (2008) Tight junctions and the modulation of barrier function in disease.
Histochem Cell Biol 130:55–70.
3. Edelblum KL, Turner JR (2009) The tight junction in inflammatory disease: Communi-
cation breakdown. Curr Opin Pharmacol 9:715–720.
4. Fanning AS, Mitic LL, Anderson JM (1999) Transmembrane proteins in the tight
junction barrier. J Am Soc Nephrol 10:1337–1345.
5. Mitic LL, Van Itallie CM, Anderson JM (2000) Molecular physiology and pathophysiol-
ogy of tight junctions I. Tight junction structure and function: Lessons from mutant
animals and proteins. Am J Physiol Gastrointest Liver Physiol 279:G250–G254.
6. Sanchez-Pulido L, Martin-Belmonte F, Valencia A, Alonso MA (2002) MARVEL: A
conserved domain involved in membrane apposition events. Trends Biochem Sci
7. Sundstrom JM, et al. (2009) Identification and analysis of occludin phosphosites: A
combined mass spectrometry and bioinformatics approach. J Proteome Res 8:808–817.
8. Marchiando AM, et al. (2010) Caveolin-1-dependent occludin endocytosis is required
for TNF-induced tight junction regulation in vivo. J Cell Biol 189:111–126.
9. Van Itallie CM, Fanning AS, Holmes J, Anderson JM (2010) Occludin is required for
cytokine-induced regulation of tight junction barriers. J Cell Sci 123:2844–2852.
10. Murakami T, Felinski EA, Antonetti DA (2009) Occludin phosphorylation and ubiqui-
tination regulate tight junction trafficking and vascular endothelial growth factor-
induced permeability. J Biol Chem 284:21036–21046.
11. Balda MS, et al. (1996) Functional dissociation of paracellular permeability and trans-
epithelial electrical resistance and disruption of the apical-basolateral intramembrane
diffusion barrier by expression of a mutant tight junction membrane protein. J Cell
12. Furuse M, et al. (1994) Direct association of occludin with ZO-1 and its possible involve-
ment in the localization of occludin at tight junctions. J Cell Biol 127:1617–1626.
13. Nusrat A, et al. (2000) The coiled-coil domain of occludin can act to organize structural
and functional elements of the epithelial tight junction. J Biol Chem 275:29816–29822.
14. Fanning AS, Anderson JM (2009) Zonula occludens-1 and -2 are cytosolic scaffolds that
regulate the assembly of cellular junctions. Ann NY Acad Sci 1165:113–120.
15. Bauer H, Zweimueller-Mayer J, Steinbacher P, Lametschwandtner A, Bauer HC (2010)
The dual role of zonula occludens (ZO) proteins. J Biomed Biotechnol 2010:402593, 10
16. Li Y, Fanning AS, Anderson JM, Lavie A (2005) Structure of the conserved cytoplasmic
C-terminal domain of occludin: Identification of the ZO-1 binding surface. J Mol Biol
17. Muller SL, et al. (2005) The tight junction protein occludin and the adherens junction
protein alpha-catenin share a common interaction mechanism with ZO-1. J Biol Chem
18. Schmidt A, Utepbergenov DI, Krause G, Blasig IE (2001) Use of surface plasmon reso-
nance for real-time analysis of the interaction of ZO-1 and occludin. Biochem Biophys
Res Commun 288:1194–1199.
19. Schmidt A, et al. (2004) Occludin binds to the SH3-hinge-GuK unit of zonula occludens
protein 1: Potential mechanism of tight junction regulation. Cell Mol Life Sci
20. Lye MF, Fanning AS, Su Y, Anderson JM, Lavie A (2010) Insights into regulated ligand
binding sites from the structure of ZO-1 Src homology 3-guanylate kinase module.
J Biol Chem 285:13907–13917.
21. Nomme J, et al. (2011) The Src homology 3 domain is required for junctional adhesion
molecule binding to the third PDZ domain of the scaffolding protein ZO-1. J Biol Chem
22. Pan L, Chen J, Yu J, Yu H, Zhang M (2011) The structure of the PDZ3-SH3-GuK tandem
of ZO-1 suggests a supramodular organization of the MAGUK family scaffold protein
core. J Biol Chem 286:40069–40074.
23. Engelman DM, Moore PB (1975) Determination of quaternary structure by small angle
neutron scattering. Annu Rev Biophys Bioeng 4:219–241.
24. Petoukhov MV, Svergun DI (2005) Global rigid body modeling of macromolecular
complexes against small-angle scattering data. Biophys J 89:1237–1250.
25. Fanning AS, Lye MF, Anderson JM, Lavie A (2007) Domain swapping within PDZ2 is
responsible for dimerization of ZO proteins. J Biol Chem 282:37710–37716.
26. Salzmann M, Wider G, Pervushin K, Senn H, Wuthrich K (1999) TROSY-type triple-
resonance experiments for sequential NMR assignments of large proteins. J Am Chem
27. Salzmann M, Pervushin K, Wider G, Senn H, Wuthrich K (1998) TROSY in triple-
resonance experiments: New perspectives for sequential NMR assignment of large
proteins. Proc Natl Acad Sci USA 95:13585–13590.
28. Takahashi H, Nakanishi T, Kami K, Arata Y, Shimada I (2000) A novel NMR method for
determining the interfaces of large protein-protein complexes. Nat Struct Biol
29. Fanning AS, et al. (2007) The unique-5 and -6 motifs of ZO-1 regulate tight junction
strand localization and scaffolding properties. Mol Biol Cell 18:721–731.
30. Reese ML, Dakoji S, Bredt DS, Dotsch V (2007) The guanylate kinase domain of the
MAGUK PSD-95 binds dynamically to a conserved motif in MAP1a. Nat Struct Mol Biol
31. Zhu J,et al.(2011) Guanylatekinasedomains ofthe MAGUKfamilyscaffoldproteinsas
specific phospho-protein-binding modules. EMBO J 30:4986–4997.
32. Raleigh DR, et al. (2010) Tight junction-associated MARVEL proteins marveld3, tricel-
lulin, andoccludin have distinctbutoverlappingfunctions. MolBiolCell 21:1200–1213.
33. Riazuddin S, et al. (2006) Tricellulin is a tight-junction protein necessary for hearing.
Am J Hum Genet 79:1040–1051.
34. Balda MS, Anderson JM, Matter K (1996) The SH3 domain of the tight junction protein
ZO-1 binds to a serine protein kinase that phosphorylates a region C-terminal to this
domain. FEBS Lett 399:326–332.
35. Elias BC, et al. (2009) Phosphorylation of Tyr-398 and Tyr-402 in occludin prevents its
interaction with ZO-1 and destabilizes its assembly at the tight junctions. J Biol Chem
36. Kale G, Naren AP, Sheth P, Rao RK (2003) Tyrosine phosphorylation of occludin attenu-
ates its interactions with ZO-1, ZO-2, and ZO-3. Biochem Biophys Res Commun
37. Raleigh DR, et al. (2011) Occludin S408 phosphorylation regulates tight junction
protein interactions and barrier function. J Cell Biol 193:565–582.
38. Rao RK, Basuroy S, Rao VU, Karnaky KJ, Jr, Gupta A (2002) Tyrosine phosphorylation
and dissociation of occludin-ZO-1 and E-cadherin-beta-catenin complexes from the
cytoskeleton by oxidative stress. Biochem J 368:471–481.
39. Du D, et al. (2010) The tight junction protein, occludin, regulates the directional
migration of epithelial cells. Dev Cell 18:52–63.
40. Yamamoto M, et al. (2008) Phosphorylation of claudin-5 and occludin by rho kinase in
brain endothelial cells. Am J Pathol 172:521–533.
www.pnas.org/cgi/doi/10.1073/pnas.1121390109Tash et al.