Molecular Determinants of the Interaction between Clostridium perfringens Enterotoxin Fragments and Claudin-3

Clostridium perfringens enterotoxin (CPE) binds to the extracellular loop 2 of a subset of claudins, e.g. claudin-3. Here, the molecular mechanism of the CPE-claudin interaction was analyzed. Using peptide arrays, recombinant CPE-(116–319) bound to loop 2 peptides of mouse claudin-3, -6, -7, -9, and -14 but not of 1, 2, 4, 5, 8, 10–13, 15, 16, 18–20, and 22. Substitution peptide mapping identified the central motif 148NPL150VP, supposed to represent a turn region in the loop 2, as essential for the interaction between CPE and murine claudin-3 peptides. CPE-binding assays with claudin-3 mutant-transfected HEK293 cells or lysates thereof demonstrated the involvement of Asn148 and Leu150 of full-length claudin-3 in the binding. CPE-(116–319) and CPE-(194–319) bound to HEK293 cells expressing claudin-3, whereas CPE-(116–319) bound to claudin-5-expressing HEK293 cells, also. This binding was inhibited by substitutions T151A and Q156E in claudin-5. In contrast, removal of the aromatic side chains in the loop 2 of claudin-3 and -5, involved in trans-interaction between claudins, increased the amount of CPE-(116–319) bound. These findings and molecular modeling indicate different molecular mechanisms of claudin-claudin trans-interaction and claudin-CPE interaction. Confocal microscopy showed that CPE-(116–319) and CPE-(194–319) bind to claudin-3 at the plasma membrane, outside cell-cell contacts. Together, these findings demonstrate that CPE binds to the hydrophobic turn and flanking polar residues in the loop 2 of claudin-3 outside tight junctions. The data can be used for the specific design of CPE-based modulators of tight junctions, to improve drug delivery, and as chemotherapeutics for tumors overexpressing claudins.

The clinical use of many promising drug candidates is impeded by unacceptable pharmacokinetics (1). The ability of a drug to pass through tissue barriers is a major determinant for its delivery. In epithelia and endothelia, the paracellular route is blocked by tight junctions (TJ). 4 Different approaches have been used to enhance transcellular drug delivery. These include the use of influx transporters, blocking of efflux transporters, or receptor-mediated endocytosis (2). Alternative approaches aim to enhance paracellular permeation of drugs by loosening the TJ (3,4). This strategy has the advantage that it could improve the delivery of structurally unrelated drugs, and the drug itself does not have to be modified. Although different TJ modulators have been described, most of these are based on surfactants or chelators (3). These often have low tissue specificity and cause severe side effects, e.g. exfoliation of cells, which irreversibly compromise the barrier functions (5,6). Fewer side effects may be obtained by more specific modulation of a molecular key component of the TJ (7).
TJ consist of transmembrane proteins, mainly the tetraspan proteins of the claudin family, as well as occludin and tricellulin (8). Other molecules associated with TJ include membranebound scaffolding and signaling proteins (9). However, claudins (Cld) are the major functional constituent of TJ (10). Claudins tighten the paracellular space, selectively for tissue, size, and charge. The tissue-specific combination of the claudin subtypes present in heteropolymers is assumed to determine the permeability properties of TJ (11). It was therefore proposed that tissue-specific drug delivery via the paracellular route would be possible by modulation of the barrier-function of claudins in a subtype-specific manner (7).
A subset of claudins, e.g. Cld3 and -4 but not -1 and -2, have been shown to be receptors for Clostridium perfringens enterotoxin (CPE) with high association constants of about 10 8 M Ϫ1 (12). CPE causes one of the most common food-borne diseases (13). It consists of two functional domains, an N-terminal region that mediates the cytotoxic effect and the C-terminal region (CPE-(184 -319)), which binds to extracellular loop 2 (ECL2) of Cld3 but not of Cld1 nor to the ECL1 of Cld3 (12). Treatment of epithelial monolayers with non-cytotoxic CPE-(184 -319) increases paracellular permeability (14). CPE-(184 -319) enhanced drug absorption in rat jejunum 400-fold relative to sodium caprate, which is in clinical use (15). Thus, CPE is a promising tool to specifically modulate claudins, the key constituents of TJ, and thereby to enhance paracellular drug delivery. In addition, some studies have suggested the use of CPE for the chemotherapy of tumors overexpressing claudins (16 -18).
Cld1 and -5 are potential targets for transepidermal and brain drug delivery, respectively (19,20). However, it has been reported that these claudins do not interact with CPE (12). Modification of CPE could enhance and/or shift its claudinsubtype specificity. Therefore, the design of CPE-based TJ modulators could permit efficient claudin subtype-specific modulation, which would also be tissue-specific modulation of TJ. To achieve this, an understanding of the molecular mechanism of the CPE-claudin interaction is a necessary prerequisite. In this study, we identify the residues within the ECL2 of Cld3 that are involved in interaction with CPE.
Peptide Arrays-Arrays were generated by spot-synthesis using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry as described (23). The AutoSpot-Robot ASS 222 (Intavis Bioanalytical Instruments AG, Köln, Germany) was used for automatic synthesis of 15-20-mer peptides on Whatman 50 cellulose membranes (C-terminal immobilization). The quality of synthesized peptides was evaluated by mass spectrometry of control spots. For binding experiments membranes were incubated with 10 g/ml GST-CPE-(116 -319) or GST in blocking buffer (Sigma) overnight at 8°C, followed by incubations with mouse anti-GST and HRP-conjugated goat anti-mouse antibody in blocking buffer for 1.5 and 1 h at room temperature, respectively. The ECL Western blotting detection reagent (GE Healthcare) was applied and chemiluminescence intensities were measured as Boehringer light units (BLU) using a Lumi-Imager (Roche Applied Science). Determined BLU values of GST-incubated SPOTs were subtracted from BLU values of corresponding GST-CPE-(116 -319)-incubated spots (⌬BLU). Background corrected ⌬BLU values were normalized to the binding of GST-CPE-(116 -319) on Cld3-spots (%Cld3).
Electrical Cell Substrate Impedance Sensing (ECIS)-Caco-2 cells were seeded into the wells of ECIS TM 8W10Eϩ electrode arrays for ECIS TM model 1600R (Applied Biophysics) at a density of 5 ϫ 10 5 cells per well and grown in a humidified CO 2 incubator with 5% CO 2 . Junctional resistance was measured every 5 min at 400 Hz frequency, as in a previous report (24). When monolayers reached maximum resistance (ϳ1500 ⍀) 10 g/ml GST-CPE-(116 -319) or GST were introduced into the medium.

Claudin-3-CPE Interaction
Measurements of Transepithelial Electrical Resistance (TEER) and Paracellular Permeation-5 ϫ 10 5 Caco-2 cells were seeded into a 24-well transwell filter (Millipore, Eschborn, Germany). TEER was determined with Endohm electrodes (Millipore). After reaching stable TEER values, cells were incubated with GST-CPE-(116 -319) or GST for 16 h. For permeation studies cells were incubated with 25 g/ml fluorescein (Sigma) in Hanks' balanced salt solution buffer on the apical side and Hanks' balanced salt solution on the basal side for 10 min. 100-l samples were removed from the basal compartment and the fluorescence units were measured using a fluorescence plate reader (Tecan, Crailsheim, Germany) to calculate the permeation coefficient.
Structural Bioinformatics and Molecular Modeling-The ECL2 of claudins was predicted by alignment of sequences of mouse (human when indicated) Cld-(1-23) with the GCG program package (GCG Wisconsin package, Accelrys Inc., San Diego, CA). A homology model for Cld3-ECL2-(134 -164) was built on the model for murine Cld5-ECL2-(135-165) as described (21). The Cld5-ECL2 model was based on part of Protein Data Bank structure 2BDV (Phage-related Protein BB2244 from Bordetella bronchiseptica). This resulted in a helix-turn-helix motif for ECL2, where the helices of the ECL2 are extensions of the predicted transmembrane helices 3 and 4 of Cld3. All manual reciprocal dockings, manipulations, and optimizations of ECL2 models were performed with the program Sybyl 7.3 (Tripos Inc., St. Louis, MO). The models were energetically minimized, using the AMBER95 force field.
Statistics-Unless stated otherwise, results are shown as mean Ϯ S.E. Statistical analyses were performed by one-way analysis of variance and followed by an unpaired Student's t test. p Ͻ 0.01 was taken as significantly different.
For the identification of amino acids involved in the CPEclaudin interaction, additional peptide arrays were performed. Because GST-CPE-(116 -319) did not bind to Cld5-ECL2 peptides (Table 1), we tested whether introduction of defined amino acids of Cld3-ECL2 into Cld5-ECL2 enables binding to CPE. In contrast to single or certain multiple substitutions including E146D, V154E, S155A, and Y158R, specifically the double substitution D149N/T151L in Cld5-ECL2 peptides facilitated the binding of GST-CPE-(116 -319) ( Table 2, upper  part). Similarly, substitution of S149N in Cld2-ECL2 corresponding to substitution of D149N in Cld5 enabled binding of GST-CPE-(116 -319) ( Table 2, lower part). The additional sub-stitution S155A in Cld2 further increased the binding. For Cld4, the double substitution M151L/A153P permitted binding of GST-CPE-(116 -319) ( Table 2,  To analyze the molecular determinants of the Cld3-CPE interaction in more detail, substitution mapping was performed. Here, peptide arrays were used that contained peptides with every possible single amino acid substitution for each position in Cld3-ECL2-(145-159) (Fig. 2). Cysteine was excluded because it leads to artificial covalent bonds during peptide synthesis. None of the substitutions at positions 145 to 147 had a considerable effect on the binding of GST-CPE-(116 -319). In contrast, every substitution in the region 148 NPLVP 152 except V151I/L/R abolished binding of GST-CPE-(116 -319). This indicates that the NPLVP motif is essential for the Cld3-CPE interaction. In addition, several substitutions at positions 154 -158 also influenced the amount of GST-CPE-(116 -319) bound. In particular, the introduction of negatively charged amino acids or proline at positions 154 -157 strongly reduced the binding of GST-CPE-(116 -319).
Interaction between Full-length Claudin-3 and CPE-(116 -319) Is Affected by Amino Acid Substitutions in ECL2-To determine whether amino acid substitutions in ECL2 affect binding of GST-CPE-(116 -319) to full-length Cld3, transfections of Cld3 in TJ-free HEK cells (21) were performed. Lysates of the cells were used for pull-down assays with GST-CPE-(116 -319) (Fig. 3). Cld3 bound specifically to GST-CPE-(116 -319) but not GST. Substitutions N148D and L150A in the ECL2 of Cld3 caused a strong reduction in the amount of Cld3 that bound to GST-CPE-(116 -319). In contrast, substitutions A154N and Q155E did not affect the interaction of full-length Cld3 with GST-CPE-(116 -319). Substitution E153V used to mimic a sequence element in Cld5 slightly reduced the amount of bound Cld3. Removal of the aromatic side chain of Tyr 147 (Y147A), which is involved in the trans-interaction between claudins (21), increased the amount of Cld3 bound to GST-CPE-(116 -319).
Binding of CPE to Claudin-3 on the Surface of Living Cells Is Affected by N148D/L150A Double Amino Acid Substitution-To analyze the binding of CPE to Cld3 in the plasma membrane of living cells, HEK cells were transfected with Cld3 wt or  20-mer peptides of the predicted ECL2 of the claudins (Cld1-16, 18, 19, 22, mouse; Cld20 human; 3rd column) were immobilized on a membrane and incubated with 10 g/ml GST (1st column) or GST-CPE-(116 -319) (2nd column). Bound CPE was visualized via mouse anti-GST and HRP-conjugated anti-mouse antibodies. Chemiluminescence intensities relative to that for Cld3 are shown (last column). Mean of n ϭ 2. Representative membranes are shown. Underlined, region (NPLVP) highly conserved between claudins that bind CPE-(116 -319). Bold, amino acids in this region that are identical to that of claudin-3.
Cld3 mutants , incubated with GST-CPE-(116 -319) or GST as a control, and the presence of bound GST-CPE in cell lysates tested by Western blots. GST-CPE-(116 -319) bound specifi-cally to Cld3-transfected but not to non-transfected cells (Fig. 4,  left). This binding was not clearly affected by single amino acid substitutions N148D or L150A but strongly reduced by N148D/ L150A double substitution in Cld3 (Fig. 4, middle). Similar results (Fig. 4, right) were obtained with GST-CPE-(194 -319), which contains a CPE fragment of which the structure has been recently solved (30).
CPE Binds to Cld3 on the Surface of Transfected HEK Cells Outside Tight Junctions-The binding of GST-CPE to Cld3transfected HEK cells was also analyzed by confocal microscopy. Cld3 was detected in the plasma membrane and intracellular compartments (Fig. 5, A-C). Marked enrichment of Cld3 at contacts between two Cld3-expressing cells was observed. This enrichment is known to correspond to the formation of TJ strands (21). No binding of GST to either Cld3 expressing or non-expressing cells was detected (Fig. 5A). In contrast, GST-CPE-(116 -319) bound specifically to Cld3 expressing cells and did not bind to non-expressing cells (Fig. 5B). Interestingly, almost no GST-CPE-(116 -319) signals were detected at the contacts between two Cld3-expressing cells, where Cld3 was strongly enriched. However, strong GST-CPE-(116 -319) signals were detected on the surface of the cells outside the contacts. Similar results were obtained with GST-CPE-(194 -319) (Fig. 5C). Cld3-N148D/L150A was enriched at contacts similarly to Cld3 wt (Fig. 5D). This indicates that the amino acid substitutions do not alter plasma membrane targeting and ability for trans-interaction of Cld3 (21). Instead, binding of GST-CPE-(194 -319) was strongly reduced. In addition, incubation of transfected HEK cells with GST-CPE-(194 -319) for 14 h resulted in internalization of CPE and Cld3 (Fig. 5E, left). This was blocked by the N148D/L150A substitution (Fig. 5E, right).

CPE-(116 -319) but Not CPE-(194 -319) Binds to Cld5-transfected HEK Cells-GST-CPE-(116 -319) not only bound to
Cld3-but also to Cld5-transfected cells (Fig. 6A). Cld3-and Cld5-YFP expression levels were detected via anti-YFP antibodies. Due to differences in the formation of SDS-resistant oligomers (31), the intensities of the Cld3-and Cld5-YFP bands were difficult to compare. Nevertheless, Cld5-YFP was expressed at least as high as Cld3-YFP, but gave a weaker signal for bound GST-CPE-(116 -319). This indicates that GST-CPE-(116 -319) binds more strongly to Cld3 than to Cld5. In contrast to GST-CPE-(116 -319), GST-CPE-(194 -319) bound to Cld3-but not to Cld5transfected HEK cells (Fig. 6B). Amino Acid Substitutions in ECL2 of Cld5 Affect the Binding of CPE-(116 -319) to Cld5-transfected HEK Cells-To identify determinants in the ECL2 of Cld5 for the binding of CPE-(116 -319), a set of Cld5-ECL2 mutants (21) was used. The plasma membrane targeting of these mutants was not affected. Transfection of most of the mutants resulted in binding of GST-CPE-  (Cld3-(154 -157)) also contains amino acids involved in the interaction. 15-mer peptides of Cld3-ECL2 were immobilized on a membrane. In the left column, all spots contain the wild type sequence (wt); the letter and number give the position of the amino acid replaced in the same line of the second column. The membrane was incubated with 10 g/ml GST-CPE-(116 -319). Bound CPE was visualized via mouse anti-GST and HRP-conjugated anti-mouse antibodies. Binding was considered to be decreased or increased if the mean chemiluminescence was increased or decreased by a factor of 4, respectively, compared with the wt (n ϭ 2). AA, amino acid; X, every amino acid except Cys and those mentioned as less or stronger. Dotted circles, wt sequence; dashed boxes, substitutions resulting in decreased binding. A representative membrane is shown.

trans-interaction
Structural Determinants of Claudin-CPE Interaction-Our homologous helix-turn-helix model for the ECL2 of Cld3 shows a strong hydrophobic area at the turn ( 149 PLVP, green in Fig.  7C) between the helices. This probably matches complementary with a hydrophobic pit on the surface of CPE formed by residues (Tyr 306 , Tyr 310 , Tyr 312 , Leu 315 , magenta, Fig. 7E). The Cld3 ECL2 residues Asn 148 and Lys 156 (blue in Fig. 7C) form loop-stabilizing hydrogen bridges. The residues (Phe 146 , Tyr 147 , and Arg 157 , yellow in Fig. 7E) point away from the potential CPE-binding motif and do not seem to be involved in CPE binding.

DISCUSSION
This study identifies amino acid residues and a sequence motif in the ECL2 of claudin-3 involved in the interaction with CPE by the use of biochemical and cellular approaches. Together with molecular modeling and literature data the results allowed us to deduce determinants of the interaction.
In the recently solved x-ray structure of CPE-(194 -319), region 290 -319 is part of a nine-strand ␤ sandwich that forms the claudinbinding domain (30). This explains our SPR results (Fig. 1) showing that the ECL2 of Cld3 interacts more weakly with GST-CPE-(290 -319) than with GST-CPE-(116 -319), because the latter includes the whole binding domain.
Low and High Affinity Binding of CPE to Full-length Claudins-GST-CPE-(116 -319) pull-down assays with lysates of Cld3-transfected HEK cells showed that substitutions N148D and L150A strongly inhibit the interaction of GST-CPE-(116 -319) with full-length Cld3 (Fig. 3). This is comparable with findings obtained by ECL2-peptide arrays. Substitutions at other positions in full-length Cld3 did not inhibit the interaction to a similar extent.
Binding of GST-CPE-(116 -319) and GST-CPE-(194 -319) to Cld3 on the surface of living HEK cells was diminished by the

Claudin-3-CPE Interaction
double substitution N148D/L150A, but not by the single substitutions N148D and L150A (Figs. 4 and 5). This difference to the pull-down assay indicates that, in addition to amino acid substitutions, detergent solubilization decreases the affinity/ avidity of the CPE-Cld3 interaction. This could be due to dissociation of multimeric claudin complexes (21) and/or destabilization of the claudin loop structure caused by solubilization.
In other studies, CPE-Cld5 interaction could not be detected (35). However, these authors used CPE-(184 -319), a short fragment similar to CPE-(194 -319), which, in contrast to CPE-(116 -319), shows no binding to Cld5 in our study either. Fujita et al. (12) reported no high affinity binding of full-length CPE to Cld5. This does not rule out low affinity binding. Taken together, the data obtained with claudin peptides, full-length claudins, and from the literature, are consistent with the assumption of high affinity binding of CPE to Cld3 and low affinity binding of CPE to Cld5.
Previously, we characterized a set of Cld5-ECL2 mutants that were targeted to the plasma membrane in a similar manner to Cld5 wt (21). To assess the CPE-Cld5 interaction in more detail, CPE binding to HEK293 cells transfected with these mutants was analyzed. In contrast to all other analyzed Cld5 mutants , FIGURE 7. Schemes of molecular interaction between CPE and the ECL2 of claudin-3. A, topology of claudins with ECL2 marked in a box. B, alignment of ECL2 of Cld3 and -5, with similarity of residues calculated with matrix blossom62 (dot ϭ weak; colon ϭ strong similarity; bar ϭ identity). Secondary structure (SecStr) according to the model, helices (H), turn (t). C, homologous helix-turn-helix model for ECL2 of Cld3 based on the fragment of PDB code 2BDV in front view. N-terminal helix, orange; C-terminal helix, cyan. The hydrophobic residues 149 PLVP (green) constituting the turn region of the loop are found to be important for CPE binding. The residues Asn 148 and Lys 156 (light blue) stabilize the turn-fold of the ECL2 by hydrogen bridges (hydrogens in cyan). Residues Phe 146 , Tyr 147 , and Arg 157 (yellow) correspond to aromatic residues Phe 147 , Tyr 148 , and Tyr 158 in Cld5, which were found to be important for trans-interaction. Heteroatoms are red (oxygen) and dark blue (nitrogen). D, according to the results, CPE cannot bind to trans-interacting claudins (left), probably due to steric hindrance, but needs a free ECL2 for binding (right). E, supposed spatial regions of interaction between binding sensitive residues (Tyr 306 , Tyr 310 , Tyr 312 , and Leu 315 from literature (36)) visualized at the CPE-(194 -319) x-ray structure (PDB code 2QUO) and the residues ( 148 NPLVP, Gln 155 ) identified in this study for Cld3.
Structural Determinants Support an Interaction of the Helixturn-helix Conformation of the ECL2 of Cld3 with CPE-The experimental binding data were combined with molecular homology modeling. Our previously generated helix-turn-helix homology model for the ECL2 of Cld5 indicates that mutations of exactly those residues that are supposed to stabilize the turn conformation led to folding defects (11). Because all of these stabilizing residues are identical (except a Asp 3 Asn exchange, which still preserves the same H-bonds) in Cld3, a comparable turn conformation could also be adapted for the ECL2 of Cld3 (Fig. 7). This is consistent with results of GST-CPE-(116 -319) binding on ECL2 peptide arrays of Cld3 found in this study. For exactly those residues forming the turn ( 148 NPLVP), almost any possible substitution abolished CPE binding (Fig. 2). Substitutions at Val 151 that preserved CPE binding were mainly hydrophobic. This is consistent with the suggested turn conformation, because Val 151 forms the hydrophobic core of the turn between the two helices and is itself not supposed to be involved in the CPE binding. The hydrophobic property on the surface of this turn portion of our helix-turnhelix model is formed by nPLvP.
In CPE, Tyr 306 , Tyr 310 , Tyr 312 , and Leu 315 have been shown to be involved in the CPE-Cld4 interaction (36,37). Similarly, we found that deletion of amino acids 310 -319 diminished binding of Cld3 and Cld4 to GST-CPE (supplemental Fig. S1). On the basis of the complementary properties and shape, direct interaction of the hydrophobic ECL2-turn with a hydrophobic pit on the surface of CPE formed by Tyr 306 , Tyr 310 , Tyr 312 , or Leu 315 (30) is very likely. This is supported by mutations Cld3-L150A, which strongly reduced the binding (Fig. 3), and additionally by the peptide array data (Fig. 2). Substitution of N148D, flanking the hydrophobic turn, strongly reduced CPE binding also (Fig. 3). This also indicates that the amino group of Cld3-N148 is involved in interaction with CPE. In addition, double substitution N148D/L150A in Cld3 diminished binding of GST-CPE-(116 -319) and GST-CPE-(194 -319) (Figs. 4 and 5). Consistently, Cld5 whose turn (DPTVP) contains no amino group and a smaller hydrophobic property than that of Cld3 (NPLVP) binds more weakly to CPE than does Cld3. CPE has been shown to bind to full-length Cld4 and Cld8 (12,14). Indeed, we found that GST-CPE-(116 -319) and GST-CPE-(194 -319) bind to full-length Cld4 (supplemental Fig. S1). In contrast, we did not observe an interaction between CPE and synthesized ECL2-peptides of Cld4 and Cld8. Similar results have been reported by others earlier. They found that CPE peptides do not bind to claudin-4 fragments without transmembrane segments (38). Strikingly, Cld4 and -8 do not contain the conserved second proline implicated in the stabilization of the loop structure (11). In addition, Cld4 is the only CPE-binding claudin that contains a methionine, instead of leucine (Leu 150 in Cld3), in the turn region. It is possible, that due to lack of the corresponding leucine and proline, the conformation of ECL2 peptides of Cld4 and -8 on the peptide mapping membrane differs more markedly from the native structure than it does for CLd3, -6, -9, or -14. Indeed, substitutions of M150L/A153P in the Cld4 ECL2 peptide enabled binding to GST-CPE-(116 -319) ( Table 2). Our results with ECL2 peptides and full-length claudins, as well as other reports (38), are consistent with the idea that transmembrane segments stabilize the native conformation of the ECL2 and, thereby, increase the affinity of the CPE-claudin interaction.
CPE Binds to the Free ECL2 of Claudins Outside of Tight Junctions-Aromatic residues in CPE (Tyr 306 , Tyr 310 , Tyr 312 ) are involved in interaction with claudins (36) and formation of an aromatic core within an ECL2-dimer is involved in the transinteraction of claudins between two opposing cells (21). Others have speculated that aromatic residues in claudin could also interact with the aromatic residues in CPE (38). But interestingly, removal of the aromatic residues (F147A, Y148A, Y158A) in Cld5 resulted in an increase in binding of GST-CPE-(116 -319) to transfected cells (Fig. 6). Similarly, substitution of Y147A in Cld3, which corresponds to Cld5-Y148A and also blocks trans-interaction, increased the amount of Cld3 pulled down with GST-CPE-(116 -319) (Fig. 3). Moreover, in our model, the aromatic residues in the ECL2 do not sterically match the aromatic residues in CPE (Fig. 7). These data indicate that residues in the turn region of the loop, but not aromatic residues in the flanking helices, are involved in the claudin-CPE interaction.
It has to be stressed that the aforementioned substitutions (F147A, Y148A, and Y158A in Cld5; Y147A in Cld3) and E159Q in Cld5 block trans-interaction and strand formation of claudins (21) as well as increase binding of GST-CPE-(116 -319). Consistently, confocal microscopy showed binding of GST-CPE-(116 -319) and GST-CPE-(194 -319) to Cld3 outside the tight junction area. These observations demonstrate that CPE binds to the free ECL2 of claudins that is not occupied by transinteraction and not incorporated in TJ strands.
Treatment of epithelial cells with CPE increases paracellular permeability. To explain this, two mechanisms have been discussed (14). Either 1) CPE binding to TJ strands leads to direct depolymerization of the claudins, or 2) CPE sequesters claudins in the plasma membrane, preventing their incorporation in the strands during the dynamic assembly of strands. Our results strongly support the sequestration mechanism and this explains our and other findings (37) in which CPE treatment needs hours for the TJ to be opened, despite high affinity interaction with claudins (12).
In conclusion, we have shown that the molecular mechanism of the CPE-claudin-3 interaction is different from the transinteraction between claudins. This improves the understanding Claudin-3-CPE Interaction JULY 10, 2009 • VOLUME 284 • NUMBER 28 of the molecular organization of TJ. In addition, the information gained is highly relevant for the design of CPE-based claudin modulators to improve drug delivery across tissue barriers (7) or treatment of tumors overexpressing claudins (16 -18).