Accepted Manuscript Title: Assembly and function of claudins: Structure-function relationships based on homology models and crystal structures Author: G.Krause J. Protze J. Piontek PII: DOI: Reference:

S1084-9521(15)00096-8 http://dx.doi.org/doi:10.1016/j.semcdb.2015.04.010 YSCDB 1761

To appear in:

Seminars in Cell & Developmental Biology

Received date: Revised date: Accepted date:

13-3-2015 28-4-2015 29-4-2015

Please cite this article as: G.KrauseProtze J, Piontek J, Assembly and function of claudins: Structure-function relationships based on homology models and crystal structures, Seminars in Cell and Developmental Biology (2015), http://dx.doi.org/10.1016/j.semcdb.2015.04.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Assembly and function of claudins: Structure-function relationships based on homology models and crystal structures

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G.Krause *[email protected], J. Protze , J. Piontek

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Leibnitz-Institut fuer molekulare Pharmakologie (FMP), 13125 Berlin, Germany

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Institute of Clinical Physiology, Charité - Universitätsmedizin Berlin, 12203 Berlin, Germany

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Abstract

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The tetra-span transmembrane proteins of the claudin family are critical components of formation and function of tight junctions (TJ). Homo- and heterophilic side-by-side (cis) and intercellular head-to-head (trans) interactions of 27 claudin-subtypes regulate tissue-specifically the paracellular permeability and/or tightness between epithelial or endothelial cells.

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This review highlights the functional impact that has been identified for particular claudin residues by relating them to structural features and architectural characteristics in the light of structural advances, which have been contributed by homology models, cryo-electron microscopy and crystal structures.

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The differing contributions to the TJ functionalities by claudins are dissected for the transmembrane region, the first and the second extracellular loop of claudins separately. Their particular impact to oligomerisation and TJ strand- and pore- formation is surveyed. Detailed knowledge about structure– function-relationships about claudins helps to reveal the molecular mechanisms of TJ assembly and regulation of paracellular permeability, which is yet not fully understood.

Keywords: Tight junction proteins; paracellular permeability; strand assembly, -disassembly; pore formation; Clostridium perfringens enterotoxin

Contents 1. Introduction, Structural segments of claudins and their functional impact

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2. Claudins: The bundle of four transmembrane helices, models and crystal structures 3. Extracellular loop 1 (ECL1) of claudins determines selective paracellular permeability versus tightness 3.1. ECL1-residues affecting paracellular permeability for solutes

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3.2. Mapping permeation and sealing sensitive positions onto ECL1 structure of Cldn15 4. Extracellular loop 2 (ECL2) of claudins

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4.1. ECL2 participates in intermolecular interaction 4.2. ECL2 homology model of Cldn5 versus crystal structure for Cldn15

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5. Interaction of ECL2 of claudins with Clostridium perfringens enterotoxin (CPE)

5.1. Critical residues for binding identified on both sides; on Cldn-ECL2 and on CPE

5.3. Modified CPE, targeting claudin subtypes

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5.2. Interacting interface between claudin and cCPE

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6. Homo- and heterophilic oligomerisation of claudins via cis- and trans-interactions

1. Introduction

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6.1. Architectural model of TJ strands in an antiparallel double row cis-arrangement

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Structural segments of claudins and their functional impact Tight junctions (TJ) consist of a number of proteins (reviewed in [1–3]), expressed in a tissue-specific manner including the blood brain barrier [4]. However the molecular mechanisms regulating paracellular sealing and permeation are still not understood. Now, for several TJ proteins structures are available such as for the extracellular domains of the junctional adhesion molecule (JAM) 1, PDB: 1NBQ, [5] and intracellular proteins such as Zona occludens protein 1 (ZO1), PDB: 2RRM, [6] and the cytosolic domain of occludin, PDB: 1XAW, 1WPA, [7] including the phosphorylation sites, PDB: 3G7C, [8] demonstrating the interactions with ZO1. As a milestone in TJ research, crystal structures for claudin-15, PDB: 4P79, [9] (Fig.1A) and claudin-19, PDB: 3X29, [10] were recently solved. The tetraspan transmembrane protein family of claudins (Cldn) are critical components for the function of TJ. On the one hand, contributions of diverse claudins, segments and individual residues thereof regulation of paracellular permeability have been revealed using heterologous expression of claudin mutants in polarized epithelial monolayers and subsequent electro physical- and permeability analyses. On the other hand, claudin-claudin interactions were analysed using TJ strand reconstitution in unpolar cells without endogenous TJs [11] and using a split-ubiquitin assay in yeast [12]. These studies have been reviewed previously [2,13,14]. Therefore, this review is focusing on the structurefunction relationships of claudins, of which 27 subtypes in human are known [15,16]. Sequence similarity analysis of claudins led to differentiation into two groups, designated as classic claudins (1-

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10, 14, 15, 17, 19) and non-classic claudins (11-13, 16, 18, 20-24) [1]. We here consider the claudins in more detail according to their differing structural components, i) the four transmembrane helices, ii) the first extracellular loop (ECL1) participating in regulation of paracellular tightening and permeation iii) the second extracellular loop (ECL2) that is involved in intermolecular interactions including binding to Clostridium perfringens enterotoxin and iv) to the contributions of the respective segments for the oligomerisation of claudins within the TJ strands. The short cytoplasmic tail of claudins is here not considered explicitly.

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2. Claudin: The bundle of four transmembrane helices-, models and crystal structures

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Little was known about structural organization of claudins during the last decade. There have been few examples where models including the transmembrane domain for claudins have been suggested solely relying on web portals for automatic modelling of protein structures [17]. More extensive molecular dynamics studies were also performed [18,19]. However, the experimental basis for these simulations was limited. A low resolution structure based on cryo-electron microscopy data of the transmembrane protein IP39 of Euglena PDB: 4P79, [20] was considered as a first experimental structural input for claudins due to its evolutionary related four transmembrane helix (TMH) bundle. Another approach used extensive chimeric mutations in the transmembrane region and identified the none conserved residues in Cldn5 C128, A132, C137, F139, I142, (D149, T151), A163, I166 and L174 to be involved in claudin folding and/or assembly of Cldn5 [21]. Combining this data with evolutionary sequence couplings [22] and stepwise comparative modelling of intramolecular interfaces to address the arrangement of the transmembrane segments in a four helix bundle led to a Cldn5 comparative model [23]. The suggested claudin subtype-specific intra- and intermolecular interfaces that are formed by conserved coiled-coil helix motifs and non-conserved residues in distinct TM positions of TMH3/4 and the ECL2 of Cldn5 contribute essentially to Cldn5 assembly into TJs. The predicted arrangement of the four helix bundle was confirmed by the recently released first crystal structure of a complete claudin molecule (Cldn15), which represents a milestone in TJ research, PDB: 4P79 [9] (Fig.1A). The TM segments of the Cldn15 structure and the Cldn5 model form a left-handed four-helix bundle exhibiting a rhombic clockwise arrangement of TM helices 1-2-3-4 with coiled-coil structures [9,23]. Three (TM-helices 1,3,4) out of the four predicted partial coiled coil transmembrane helices agreed in fold and conformation with the crystal structure (Fig.1C). This crystal structure enable the generation of models of more precise helix arrangements for other claudins [23]. Apart from the four TM-helices, the two extracellular loops showing structural segments on their own, are linked via a connecting beta sheet fold (Fig.1B).

3. Extracellular loop 1 (ECL1) of claudins determines selective paracellular permeability versus tightness Claudins can subtype-dependent either completely tighten the paracellular cleft for solutes and H2O or can form paracellular pores. It has been recognized very early that the larger first extracellular loop

ECL1 (∼50 amino acids containing a conserved motif W-G/NLW-C-C including a disulphide bridge

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[24]) is involved in barrier function, enables permeability for small solutes [25] and determines the charge- [26] and size selectivity [27] of the pore. 3.1. ECL1-residues affecting paracellular permeability for solutes

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Permeation-sensitive ECL1-residues are reported for a number of pore-forming claudins. In Cldn10a R33 and R62 play a role in Cl- selectivity [28]. In human Cldn15, D55 and E64 (D55 and D64 in + murine Cldn15) determine charge selectivity [29], in Cldn16 D104, D105 and E119 contribute to Na 2+ selectivity [30] and H71, L75, G128 as well as R146 are involved in mediating paracellular Mg permeability [31]; For Cldn17 K65 was shown to be essential for Cl selectivity [32]. A conserved aromatic residue confers cation selectivity in Cldn2 (Y67) and Cldn10b (F66) [27]. Pore-relevant ECL1-residues have been intensively characterized for Cldn2. D65 was identified as an electrostatic interaction site for sodium, I66 was also found to be exposed to the lumen of the pore center, H57 was indicated to be located at a pore vestibule and Y35 suggested to be located at the protein surface outside the pore [33–36]. Comprehensive cysteine scanning mutagenesis in MDCK I cells treated with thiol-reactive reagents identified and mapped further pore-lining ECL1-residues of Cldn2: T32, G45, S47, M52, T56, T62 and D65-S68 [37].

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For TJ sealing claudins, several residues have been identified, which are responsible for sealing or where mutations lead to increased ion permeability. For Cldn4 K65 was reported to be crucial for + sealing against Na [29] and to be involved in a proposed channel function [38], dependent of the cell type. For Cldn7 D38 and E53 are suggested to contribute to the Cl barrier [39]. Additionally N44 in Cldn3 was found to prevent trans-interaction with Cldn4 [40]. The creation of single point ECL1 chimeras, replacing residues of sealing Cldn1 by corresponding residues of the pore-forming Cldn2, showed that E48K and S53E reduced TER and increased ion permeability [41]. This study suggested that S53 and potentially D68 in Cldn1 are involved in sealing of the paracellular cleft and that chargeunselective pores may be induced by S53E indicating that much more position are involved in charge selective pore formation [41].

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3.2. Mapping permeation and sealing sensitive positions onto ECL1-structure of Cldn15 The Cldn15 crystal structure revealed for ECL1 a beta sheet structure that is formed by four beta strands 1-4 [9] (see Fig.1B). We here mapped the positions of experimentally identified permeationsensitive residues from pore-forming claudins (Cldn15, -10a, -10b, (-16, mainly pore contributing), -17) and sealing claudins (Cldn1, -3, (-4, mainly sealing), -7) onto the ECL1 structure of Cldn15 (Fig.2A). The above mentioned permeation-sensitive residues of ECL1 are summarized and highlighted in figure 2B and assigned to the corresponding positions in the Cldn15 structure. Position 63 is very important, since it was identified to be critical for permeation in Cldn2, -10a, -17 and even for sealing in Cldn4. Hits in two different claudins are observed for numerous ECL1 positions corresponding to 34 (not visualized in Fig.2A), 43, 51, 55, 64, 65 and 66 in Cldn15. Moreover, positions 63, 51 and 46 are spatially assembled inline at one side of the beta sheet in Cldn15 (Fig. 2A). Since the residue properties of position 46 are complementary correlating to that of position 51 (Fig. 2B), a somehow related interaction is possible and therefore also highlighted in figure 2A. Due to the numerous experimental data of Cldn2 for ECL1 we mapped the positions in figure 2A for clarity without the data from Cldn2. Subsequently, we build a comparative ECL1-model for Cldn2 (Fig.2C) based on Cldn15 [9] and Cldn19 [10] and visualized separately those side chains, for which permeation-sensitive effects were observed in Cldn2 (Fig.2B).

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Residue D65 (corresponds to position 63 in fig 2 A,B) is mapped at the most inner position on one particular side of the beta sheet. It was previously shown to be an ion interaction site in the pore centre, whereas H57 and Y35 (boxed in Fig. 2C) mapped on outer positions of the beta sheet were previously suggested to be located at a pore vestibule and at the protein surface outside the pore, respectively [34,36]. At least D65 and H57 point to the same side of the beta sheet. According to a model for an oligomeric Cldn15 pore by Suzuki et al. [42], this particular side of the beta sheet is suggested to line the pore as part of a half pipe-like architecture (dashed blue line in Fig. 2C,D) However, for Cldn2 numerous determined pore-sensitive residues that have been accessible for thiol probes are distributed also at the rear side of the beta sheet in the model (Fig. 2C). Thus it remains unclear whether both sides of the beta-sheet contribute directly or one side indirectly to pore lining and architecture. Moreover, visualizing those residues in the Cldn15 structure (Fig.2D) that correspond to the permeation-sensitive residues in Cldn2, it is noticeable that the only positions where the residue properties are coincident in both pore-forming claudins are F65/Y67 and few hydrophilic residues (magenta) located at the most outer beta sheet positions (oval in Fig.2D). This indicates that the architecture of the two cation-pores might be at least partly different in Cldn15 and Cldn2. A 27-amino acid peptide corresponding to a portion of ECL1 (Cldn1 (53-80)) was identified to reversibly interfere with epithelial barrier by associating with the tightening Cldn1 and occludin [43]. This was taken on by studying similar synthetic peptides and recombinant constructs of ECL1. For ECL1 a two stranded β-sheet binding surface was suggested by an automatic model building procedure [44], which is to some extent in agreement at least for two of the four ECL1-beta-strands in the crystal structure of Cldn15 [9]. The already mentioned model of the oligomeric Cldn15 pore suggested cis-interaction between the ECL1 beta-sheets [42] and reflects some, but not all of the above identified permeation- sensitive claudin residues [37] and of the claudin residues involved in cisinteractions [36,42]. Taken the data together, for the ECL1 beta-sheet it is conceivable that in sealed TJ, it is tightly interacting on one hand side-by-side with the neighbour (cis) and/or on the other hand head-by-head with ECL1 from the other cell side (trans). However, these complicated multiple interactions cannot so easily be drawn from the available crystal structure of the pore forming Cldn15 structure. Therefore, further studies are necessary to understand the structural difference between sealed and permeable TJs and to reveal the pore architecture.

4. Extracellular loop 2 (ECL2) of claudins 4.1. ECL2 participates in intermolecular interaction

The second extracellular loop (ECL2) usually has ∼25 amino acids, but fewer in Cldn11 and more in Cldn18. It was suggested that the ECL2 may associate with itself and possess an anchoring function by trans-interactions across the paracellular space towards claudins in the opposite cell [11]. Cisinteractions of ECL2 with the neighbouring claudin are concluded from the observed crystal packing for Cldn15 [9]. Irrespective from the possibility that the observed crystal packing might be artificial, the analysis in the native environment is complicated by the complex composition of TJs and the numerous protein-protein interactions (PPIs) within the heteromeric TJ. Therefore, in cellular assays it

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4.2 ECL2 homology model of Cldn5 versus crystal structure for Cldn15

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is a challenge to distinguish between direct and indirect effects caused by claudin amino acid substitutions. Reconstitution of TJ strands by expression of claudin- and TAMP constructs in cells lacking endogenous TJs [11,40,45,46] reduces the complexity of the molecular composition of TJ strands, strongly reducing the amount of protein-protein interactions (PPIs) within the strands, which have to be considered. This approach combined with homology modelling of the ECL2 revealed residues conserved within classic claudins to be critical for claudin folding and/or intermolecular interaction resulting in TJ strands [11].

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A homology model of Cldn5-ECL2 based on the structural fragments from 2BDV (hypothetical protein BB2244, Bordetella bronchiseptica) suggested a helix-turn-helix conformation extending the transmembrane helices 3 and 4 at the extracellular phase (Fig.3) [47]. The turn conformation is characterized by a highly conserved proline (position P). This model fitted best to the experimental data for Cldn5 and was chosen for further analysis. The conformation is highly consistent with the observed fold defect phenotype of Cldn5 mutants D149A, P150A, V152A, P153A and K157A. The model explained also the TJ strand-deficiency of most of the mutants for Cldn5 (F147A, Y148A, Q156E, Y158A, E159Q, L160A) and Cldn3 (Y147A) [11,48] and inhibition of Cldn5 tightening function by the ECL2 mutations [49]. Comparison of this earlier Cldn5 model and the recently solved Cldn15 structure [9] revealed striking similarities but also some differences. For the Cldn5-ECL2 model [47], backbone and side chain orientation of the predicted helix-turn-helix structure agrees to 80 %, with the ECL2 conformation of the Cldn15 structure [9]. This is manifested by a pairwise fit of the back bone atoms of the N-terminal

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helices and the turn region (between P-1 and P+3) for the Cldn5-ECL2 model (blue) and the Cldn15 structure (yellow) that demonstrates a high similarity and reveals a root mean square deviation (RMSD) of 0.7 Å (Fig. 3). Only the C-terminal short beta-strand prior transmembrane helix 4 that is intramolecular interacting with ECL1 was not predicted. However, the sequence between Cldn5 and Cldn15 differ in this region. Cldn15 is missing one residue. Nevertheless, the following lysine in position P+7 (K157-Cldn5, K155-Cldn15) is stabilizing the turn conformation by forming a H-bond towards the backbone of Asp in position P-1, prior the highly conserved proline, which is also in full agreement with the crystal structure of Cldn15 [9].

5. Interaction of ECL2 of claudins with Clostridium perfringens enterotoxin (CPE) Clostridium perfringens enterotoxin (CPE) is, so far, the only natural extracellular ligand for claudins. It is one of several toxins produced by C. perfringens and causes the gastrointestinal symptoms of C. perfringens type A food poisoning (extensively reviewed in [50–52]). The C-terminal domain of CPE (cCPE) with known structure PDB: 2QUO, [53], containing the claudin binding site [54,55], is by itself not cytotoxic and increases the paracellular permeability for solutes [56,57]. Crystal structures of full length CPE are also known PDB: 2XH6, 2YHJ, [58]; PDB: 3AM2, [59]. Initially, Cldn3 and Cldn4 were described as CPE-receptors [60,61] and only later reclassified as members of the claudin family [15]. Soon after, it was shown that CPE binds to the ECL2 of distinct claudins [62] and that binding of the non-cytotoxic cCPE (CPE 184-319) removes specific claudins from TJ-strands [56]. In several studies Cldn1, -2, -3, -4, -6, -7, -8, -9 and -14 were determined as CPE binding (CPE-receptors), Cldn1 and Cldn2 showed only very weak binding [56,62–65]. However, for

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Cldn5, Cldn10-13 as well as for Cldn15-18 and 20-24 no CPE binding (none CPE-receptors) was detected [52,63]. 5.1. Critical residues for binding identified on both sides; on Cldn-ECL2 and on CPE

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On the CPE side peptide studies [66] and mutagenesis studies revealed several residues that are critical for the Cldn-CPE interaction such as Y306, Y310, Y312 and L315 [67–69]. According to the surface representation of the cCPE crystal structure, these residues frame on two sides a potential Cldn-binding pocket [64] formed by two pits (triple-Tyr- and triple-Leu-pit) of different properties and size [70]. On the claudin side peptide mapping and mutagenesis studies [63] showed the importance of certain residues in the middle of Cldn-ECL2 for Cldn / cCPE interaction leading to the identification of a CPE(P-1) (P) (P+1) (P+2) (P+3) P L/M/V binding motif (N/D V/T P/A/D ) characterized by a highly conserved proline [52,63,71]. Particularly the residues at positions P-1 prior and P+1 after this proline were found to have a major influence on the interaction with cCPE [63–65].

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5.2. Interacting interface between claudin and cCPE

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Combination of the cCPE crystal structure with the ECL2- model for classic claudins and extensive mutagenesis studies, suggested a first detailed Cldn/cCPE interaction model for Cldn3 and Cldn4 [70], (P+1) by functional linking the position L of Cldn-ECL2 to Y306 in the triple Tyr-pit of cCPE and thus defining the orientation of the binding-sensitive ECL2-turn region alongside the binding pocket of cCPE (Fig. 4A). After the release of the Cldn15 structure this interaction model was refined for Cldn3 and cCPE, where the difference is a short beta-strand instead of a helix at the C-terminal side of the ECL2, which had no influence on the binding interface of the ECL2-turn region between P-1 and P+3 [71] (orange sticks in Fig.4B). This model consequentially hinted that also the ECL1 (sand in Fig.4 B) potentially interacts with CPE. The already in 2012 predicted placement and orientation of the Cldn-ECL2-turn region in the Cldn/cCPE interaction model [70] was confirmed by the 2015 released crystal structure of Cldn19/cCPES313A complex PDB: 3X29, [10] (Fig.4 C) where position P+1 of the ECL2 is located in the triple-Tyr pit of cCPE (Fig.4 D). This important structural advance for understanding claudin interactions revealed that apart the turn region of ECL2 also the ECL1 participates and enlarges the hydrophobic surface for the interaction with cCPE. Unfortunately, the low resolution of the cCPE-Cld19 crystal structure by 3,7 Å does not allow explicit interpretation of H-bond interactions, thus the role of claudin position P-1 whether Asn or Asp stabilizes the claudin turn conformation [71] or interacts with cCPE [10] remains unclear. In 2014 a crystal structure of a modified ECL2 peptides from Cldn2 in complex with full length CPE was released (PDB: 3ZJ3), where placing and orientation of the peptide within the binding pocket was generally in agreement with both the beforehand suggested interaction model [70] and the later on released crystal structure of a complete Cldn19 complexed with cCPE [10]. A second CPE complex structure with bound short Cldn2-ECL2- peptide (YNPLVPDAM) was released PDB: 4P5H (resolution 3.34 Å) [72], showing an opposing orientation of the ECL2-fingerprint sequence NPLVP in the CPE binding pocket. There might be different reasons. Short peptides isolated from a protein particularly when modified can adopt conformations different from the native protein. 5.3. Modified CPE targeting claudin subtypes

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The development/optimization of CPE/cCPE variants with changed and tailored specificities to Cldnsubtypes, led by phage display with cCPE-S305P/S307R/S313H to a broad-specific claudin-binder [73] and by combining structural bioinformatics and mutagenesis to cCPE-variants, with preferred interactions either with Cldn3 (cCPE-L223A/D225A/R227A) or Cldn4 (cCPEL254A/S256A/I258A/D284A) [70]. Although Cldn5 is a none CPE-wt receptor, the latter approach enabled the design of a cCPE-variant (cCPE-Y306W/S313H) with shifted specificity towards Cldn5 [71]. Such cCPE-variants as well as the respective full-length CPE variants have the potential to target distinct claudin subtypes with high affinity as specific modulators for pharmacological interventions.

6. Homo- and heterophilic oligomerisation of claudins via cis and trans-interactions

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Although crystal structures of Cldn15 and Cldn19 are now available, the mechanism of TJ assembly still remains largely unclear. On the one hand, intracellular interaction of claudins with ZO proteins is necessary to stabilize endogenous TJs [74]. On the other hand, claudins are able to form TJ strands on their own in unpolar cells, independent of other TJ-associated proteins [45]. Although contribution of additional cellular factors (proteins, lipids) cannot be excluded, the assembly of claudins into TJ strands probably depends directly on the following claudin-claudin interactions: (I) intramolecular folding within the membrane and intermolecular assembly by (IIa) co- or posttranslational cisoligomerisation (within one membrane) and (IIb) trans-interaction (between opposing plasma membranes) which mediates the formation of polymeric strands [11,75]. Most epithelial and endothelial cells express an assortment of different claudins. Heteropolymeric TJ strands are formed by homophilic (between same claudins) and heterophilic interactions (between different claudins) [40,45,48]. However, not all claudins are compatible with each other. For instance, Cldn1 interacts in trans with Cldn3 but not with Cldn2; Cldn16 and Cldn19 interact in cis but not in trans (for an overview [1,2]).

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Until recently, mechanistic analysis of TJ strand assembly was limited by the shortage of structural information of claudins or homologous proteins. Euglena IP39, a member of the PMP-22/ EMP/MP20/claudin superfamily, shares 20% sequence similarity and the ability to polymerize (but not TJ formation) with claudins. Its three-dimensional structure was determined by cryo-electron microscopy of two-dimensional crystals and considered as a proxy for claudins and their potential polymer architecture [20]. However, the resolution of ~10 Å shows only the shape of the protein. Later on it turned out that the suggested arrangement of the 4-helix bundle for IP39 based on the LTC4 Synthase is completely different than that in the crystal structures of Cldn15 [9] and Cldn19 [10]. 6.1. Architectural model of TJ strands in an antiparallel double row cis-arrangement The recently, suggested architectural model of TJ strands is based on oligomeric structures in Cldn15 crystals [42]. The model contains an antiparallel double row cis-arrangement of Cldn15 protomers with two different cis-interfaces between two neighboured protomers. One interface (linear cis), is suggested between ECL2 and a short extracellular helix (ECH) of ECL1 and indicated by the linear arrangement of claudin protomers in the Cldn15 crystal packing [9] (model scheme Fig.5). Strikingly, this interface contains ECL2-residues (F146, F147, E157, L158) of which the corresponding residues in Cldn5 and in Cldn3 have previously been shown to be strongly involved in TJ strand formation [11,48]. Also for Cldn15, involvement of the corresponding F146, F147 in ECL2 was shown by freeze fracture EM [9]. In addition, M68A and M68E in ECH of Cldn15 blocked strand formation [9]. However,

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these findings support but do not prove that this ECL2 and ECH regions interact directly in cells. It is unclear whether this linear cis interface observed in the crystal packing is part of native TJ-stands. It could be artificial similar as other protomer interfaces found in the Cldn15 (PDB: 4P79) and other crystals. Alternatively, the ECL2 could participate, for instance, directly in trans-interaction as suggested earlier for Cldn5 [11].

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In sum, the data obtained with Cldn3, -5, and -15 mutants together with the Cldn15 crystal structure (9-10) ,E, L/I/M/F) on one side of the ECL2, and homology modelling strongly suggest the motif F, Y/F, x which is conserved among classic claudins to be essential for an intermolecular interaction leading to TJ strand formation.

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The second cis-interface (face-to-face) of the double row model is suggested to be formed through interactions between the edges of the β-sheets, via two β4-strands of ECL1 forming an extended beta sheet and a half pipe-like architecture [42] (model scheme Fig. 5). This interface is supported by crosslinking data obtained with mutants of another pore-forming classic claudin, Cldn2, expressed in well characterized stable MDCK cell lines [24,34]. In addition, the interface is consistent with cysteine crosslinking data, which Suzuki et al. obtained with Cldn15 mutants. However, in contrast to the Cldn2 analysis, the Cldn15 mutants were transiently expressed in sparsely cultured HEK293 cells, where the mutants could not assemble into TJs [42]. Hence, it is unclear, whether the detected dimers of the respective mutants (e.g.N61C) are of relevance for assembled TJs.

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Furthermore, it has to be noted that the claudin cis arrangement suggested by Suzuki et al. does not contain intermolecular interaction of the TMHs of the claudins, which is rather unlikely since such hydrophobic interactions are thermodynamically favoured and indicated by experimental data [21,26,76].

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Suzuki et al further suggested joining of the “half pipe” architecture of the two claudin cis-double rows between opposing cells. This would result in formation of a β-barrel-like structure of the pore with the side chains pointing into the interior of the pore [42]. However, such an arrangement shows very tight head-to-head packing in the regions of missing residues in extracellular loops ECL1 (9 residues missing) and ECL2 (2 residues missing), which when inserted lead subsequently to massive clashes, partial disturbance of the shown arrangement and possibly even of the suggested interesting pore architecture. In sum, other arrangements or at least variations of the model of TJ strands suggested by Suzuki et al. are conceivable. Nevertheless, the model provides a very valuable initial working hypothesis about the architecture of TJ strands, which can be tested in further studies. In particular, the fact that the antiparallel double row cis-arrangement of claudin protomers leads to symmetry and size consistent with the linear claudin polymeric TJ strands visualized by freeze fracture EM [42,45] is of interest. Very recently, it was proposed that the crystal structure of mCldn19 in complex with cCPE provides structural insight into aspects of tight junction (dis)assembly [10]. In contrast to the Cldn15 structure, the Cldn19-cCPE complex shows no density of a short extracellular helix (ECH) in the ECL1 proposed to be critical for claudins to assemble into TJ strands. The authors suggest that an ECH displacement induced by cCPE-binding to linear claudin polymers within one membrane may thus cause cCPE– mediated disassembly of TJs. However, there is until now no direct evidence for such a (single) strand intermediate within one membrane. In contrast, in (paired) TJ-strands cCPE-binding should be blocked since the cCPE-interacting ECL2 and ECL1 residues are very likely at least partly involved in intermolecular interfaces and sterically not accessible for the bulky cCPE. Thus, cCPE binds rather to

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claudin protomers, which are not part of TJ-strands, thereby preventing claudin incorporation into TJs and in turn destabilizing them over the time, as suggested previously [63].

Acknowledgment

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The Authors gratefully acknowledge support by DFG KR1273/3-2.

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In sum, the experimental and modelling data indicate that ECL1, ECL2 and TMHs together contribute to the formation of heteropolymeric TJ strands by homophilic and heterophilic cis- and transinteractions between claudins at cell-cell contacts. The recent advances in claudin crystallization provided ground-breaking insight in the structure of claudin protomers and enables improved structure function studies to elucidate the molecular mechanism of TJ formation.

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Figure legends

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Figure 1: A) Crystal structure of Cldn15, B) the four beta strands (1-4) of ECL1 (olive), together with the beta– strand (5) in the C-terminal part of ECL2 (lime), form a common beta-sheet which structurally links ECL1 and ECL2. C) Comparison of the transmembrane four helix bundle of Cldn15 crystal structure (yellow) [9] and suggested coiled coil helix model for TMH bundle of Cldn5 (blue) [23].

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Figure 2: Permeation- and sealing -sensitive residues of ECL1. A) Cldn15-ECL1 structure, mapped positions (spheres) of pore-sensitive residues for diverse claudins (without Cldn2), colors correspond to B. B) Tabulated claudin-residues identified as permeation-sensitive (bold black) or involved in sealing (bold blue) are assigned to the corresponding position in Cldn15 structure in A. C) Homology model of Cldn2 based on crystal structures of Cldn15 [9] and Cldn19 [10]; close up view along the beta-sheet of ECL1 (dark grey). Visualized are hydrophilic (magenta) and hydrophobic (green) side chains of permeation-sensitive residues for Cldn2. D65, interaction site for sodium, [36] (corresponds to position 63 in A, B) is located at inner position whereas .H57 (located at pore vestibule) and Y35 (located at protein surface) [34] are located at outer positions. D65 and H57 are located at the same side of the beta sheet. Orientation of side chain of Y35 is unclear, since the corresponding residue in Cldn15 appears to be flexible and showed no electron density in the Cldn15 crystal structure (4P79 [9]). The orientation of the side chain of the corresponding residue in the crystal structure of cCPE bound Cldn19 [10] may differ from the side chain orientation in free or trans- and cis- interacting claudins. Since Cldn2 pore-sensitive residues are distributed at both sides of the beta sheet, it remains unclear whether both sides contribute directly or one indirectly to the pore architecture. The beta sheet is suggested [42] to form a half pipe (dashed blue line) contributing to the pore architecture (see also Fig. 5). D) Cldn15 structure; shown are residue positions corresponding to the permeation-sensitive residues in Cldn2, it is noticeable that the only positions of coinciding properties in both pore forming claudins are F65/Y67 and few hydrophilic residues (magenta) (oval).

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Figure 3: Close up view: Superposition of ECL2 for both the Cldn5 ECL2 model [47] (blue) and the Cldn15 structure [9] (yellow) revealed identical conformation for the N-terminal helix and the following positions between P-1 to P+3. The C-terminal part is different. In that respect, it is noticeable that the Cldn15 sequence is missing one residue here (see sequence alignment). Nevertheless, in the Cldn15 structure and the Cldn5 model the highly conserved Lys in position P+7 interacts with its side chain identically with the backbone in P-1 stabilizing the turn conformation via H-Bond.

Figure 4: Claudin-cCPE interaction interface: A) Cldn4 helix-turn-helix ECL2 (cyan) interaction model with cCPE surface representation. Position P+1 of Cldn-4 is located in the triple-Tyr pit of cCPE [70]. B) Refined Cldn3 helixturn-beta-strand ECL2 (orange sticks) interaction model with cCPE [71] Cldn3-ECL1 (sand) and -2 (orange) are based on Cldn15 crystal structure [9]. C) Crystal structure of Cldn19 (beige) and cCPE interaction (grey surface) [10] D) its close up view confirmed the orientation of position P+1 in ECL2 within the triple-Tyr pit of cCPE. Apart the turn region of ECL2 (beige sticks) also ECL1 is clearly interacting with cCPE.

Figure 5: The oligomeric Cldn15 model (A) suggested by Suzuki [42] and its simplified scheme (B) for the antiparallel double row cis-arrangement of Cldn15. The two different cis-interfaces between the extracellular loops of neighbouring protomers contain i) a linear cis interface between ECL2 (lime) and a short extracellular helix (ECH) of ECL1 (orange) and ii) a face-to-face cis-interface between the edges of the ECL1-β-sheets from two protomers (orange and blue). They form an extended beta sheet that shapes a half pipe participating in the pore architecture (red arrows). The model lacks intermolecular interactions between transmembrane domains (TMD) of the claudin protomers.

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