Multiscale modelling of claudin-based assemblies: A magnifying glass for novel structures of biological interfaces

Abstract

Claudins (Cldns) define a family of transmembrane proteins that are the major determinants of the tight junction integrity and tissue selectivity. They promote the formation of either barriers or ion-selective channels at the interface between two facing cells, across the paracellular space. Multiple Cldn subunits form complexes that include cis- (intracellular) interactions along the membrane of a single cell and trans- (intercellular) interactions across adjacent cells. The first description of Cldn assemblies was provided by electron microscopy, while electrophysiology, mutagenesis and cell biology experiments addressed the functional role of different Cldn homologs. However, the investigation of the molecular details of Cldn subunits and complexes are hampered by the lack of experimental native structures, currently limited to Cldn15. The recent implementation of computer-based techniques greatly contributed to the elucidation of Cldn properties. Molecular dynamics simulations and docking calculations were extensively used to refine the first Cldn multimeric model postulated from the crystal structure of Cldn15, and contributed to the introduction of a novel, alternative, arrangement. While both these multimeric assemblies were found to account for the physiological properties of some family members, they gave conflicting results for others. In this review, we illustrate the major findings on Cldn-based systems that were achieved by using state-of-the-art computational methodologies. The information provided by these results could be useful to improve the characterization of the Cldn properties and help the design of new efficient strategies to control the paracellular transport of drugs or other molecules.

Keywords: APBS, adaptive Poisson-Boltzmann solver; BBB, blood-brain barrier; CAPRI, critical assessment of prediction of interactions; CG, coarse grained; CLDN, claudin; CNS, central nervous system; CV, collective variable; Claudin; Coarse grained molecular dynamics simulations; EA+, ethyl ammonium; ECH, extracellular helix; ECL, extracellular loop; FE, free energy; FFEM, freeze-fracture electron microscopy; FRET, fluorescence resonance energy transfer; Free energy calculations; GPU, graphics processing unit; HB, hydrogen bonds; ICL, intracellular loop; MA+, methylammonium; MD, molecular dynamics; MDCK, Madin-Darby canine kidney; MFPT, mean first passage time; Molecular dynamics simulations; NMR, nuclear magnetic resonance; PAGE, polyacrylamide gel electrophoresis; PANEL, protein association energy landscape; PDB, protein data bank; PMF, potential of mean force; Protein-protein molecular docking; SF, selectivity filter; STED, super-resolution stimulated emission microscopy; TEA+, tetraethylammonium; TEER, transepithelial electric resistance; TEM, transmission electron microscopy; TJ, tight junction; TMA+, tetramethylammonium; Tight junctions; US, umbrella sampling; WT, wild type; X-RD, X-ray diffraction; cCPE, C-terminal fragment of the Clostridium perfringens enterotoxin.

Graphical abstract

Fig. 1

The epithelial junctional complexes. (A) Schematic representation of epithelial intercellular junctions. (B) Illustration of the major junctions (left) and of the main associated proteins (right). (C) Schematic representation of the TJ strand along the basolateral membrane. Parts of the figure were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).

Fig. 2

Claudin folding and multiple sequence alignment between classic homologs. (A) 2D cartoon representation of the Cldn structure. TM helices are shown with orange cylinders, ECH is represented with the yellow cylinder, $\mathrm{\beta }$-strands are indicated with green arrows. The conserved disulfide bond between $\mathrm{\beta }$1- $\mathrm{\beta }$2 is represented with the purple line. The location of the conserved motifs and the variable regions (V1, V2) are indicated. (B) Multiple sequence alignment of common classic Cldns. The motifs are highlighted. Amino acid letters are colored according to their polarity: apolar in red, polar in green, acidic in blue and basic in magenta. Symbols at the bottom of the alignment identify conserved residues: “*” for sequence identity, “:” for sequence similarity and “.” for semi-conserved residues. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

The claudin-15cis-linear interface. (A) Structure of the Cldn15 monomer. The negatively charged surface is shown in the region including Asp55, Asp64 and Glu46. The hydrogen bond between Pro149 and Lys155 in ECL2 is reported. (B) cis-linear arrangement of Cldn15 monomers stabilized by the interaction between the Met68 residue in each subunit and the crevice formed by Phe146, Phe147 and Leu158 in the ECL of the adjacent protein. Electrostatic potential surface was computed with the Adaptive Poisson-Boltzmann Solver software . Isosurfaces are drawn with a red-white-blue color scale ranging from −5.0 (red) to + 5.0 (blue) kT/e. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Experimental claudin structures. (A) Cldn15 (PDB ID: 4P79). (B) Cldn3 WT (PDB ID: 6AKE), P134G (PDB ID: 6AKF), P134A (PDB ID: 6AKG). (C) Cldn4 (PDB ID: 5B2G). (D) Cldn9 (PDB ID: 6OV2). (E) Cldn19 (PDB ID: 3X29). Missing fragments in the ECL and ICL domains are indicated.

Fig. 5

Multimeric claudin-15 arrangement introduced in Ref.. (A) Paracellular space with a sequence of identical pore-like structures, viewed from the apical side. (B) cis face-to-face interface. (C) cis-linear configuration. The key interaction between Met68 of a subunit and the Phe146, Phe147 and Leu15 residues belonging to the adjacent monomer is highlighted. (D) Single-pore model. Missing residues are indicated.

Fig. 6

Self-assembly claudin-5 coarse-grained molecular dynamics simulations performed in Ref.. (A) Snapshots of Cldn5 (red) cis self-assembly process in a lipid bilayer (blue) at different time frames. (B) Frontal and apical view of the five putative Cldn5 dimers isolated from the CG MD simulations. Reprinted with permission from Ref. . Copyright 2016, American Chemical Society. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

The back-to-back interface and the Pore II configuration. (A) Dimer B structure from frontal and apical views. The leucine zipper between TM2 and TM3 is shown. (B) Pore II configuration resulting from the trans-interactions of two dimers B.

Fig. 8

Claudin-15 single-pore and double-pore models. Superposition of the initial (pink ribbon) and final (orange ribbon) configurations of the single-pore (A) and double-pore (B) models of the Cldn15-based TJs. Reprinted with permission from Ref. . Copyright 2017, licensed under CC BY. (C) PMF (FE) profiles of the ionic permeation through the Cldn15-based channel. (D) Kinetic estimation of the mean first passage time of ions through the pore. Reprinted with permission from Ref. . Copyright 2018, American Chemical Society. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 9

Pore radius profiles for single-pore configurations of claudin-15. Positions of the charged pore-lining residues are indicated along the pore axis. The protomer of belonging of each Asp55 (blue line) and Asp64 (red line) is indicated with the letter “P”. Reprinted with permission from Ref. . Copyright 2017, licensed under CC BY. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 10

Novel cis-interfaces predicted for claudin-15. Binding poses are based on blind docking and they resemble the rotation of monomers in long strands. Reprinted with permission from Ref. . Copyright 2018, licensed under CC BY.

Fig. 11

Resistor array model of theclaudin-2strand. Three-strand TJ model represented as alternating in series and in parallel resistors in the proximity of the recording electrode. Ref. . Copyright 2017 by John Wiley & Sons, Inc. Reproduced with permission of John Wiley & Sons, Inc. in the format of Journal/magazine via Copyright Clearance Center.

Fig. 12

Pore I paracellular profile of claudin-2. (A) Lateral view of the pore-lining residues pointing towards the cavity center. The excluded molecular volume is shown in purple. (B) Pore diameter profile along the pore axis. Reprinted with permission from Ref. . Copyright 2018, American Chemical Society. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 13

Claudin-4 tetrameric models and free energy profiles. Lateral views of Pore I (A) and Pore II (B). Polar residues of two opposing protomers are shown and colored according to their charge: blue for basic residues, orange for acidic residues and green for neutral residues. FE profiles of ions and water translocation through Pore I (C) and Pore II (D). FE landscapes were reproduced with the Matplotlib library and colored with the ‘PRGn’ colormap, ranging from −10.0 (purple) to + 10.0 (green) kcal/mol. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 14

Claudin-5 tetrameric models and free energy profiles. Lateral views of Pore I (A) and Pore II (B). Monomers are indicated with different coloring. Polar residues of two subunits are shown and colored according to their charge: blue for basic residues, orange for acidic residues and green for neutral residues. FE profiles of ions and water translocation through Pore I (C) and Pore II (D). FE landscapes were reproduced with the Matplotlib library and colored with the ‘PRGn’ colormap, ranging from −10.0 (purple) to + 10.0 (green) kcal/mol. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 15

Superposition of claudin-4 and claudin-5 pore models. Superposition of Cldn4 (orange) and Cldn5 (blue) tetramers in the Pore I (A) and Pore II (B) configurations. Pore-lining residues are shown as cyan and orange sticks for Cldn5 and Cldn4, respectively. Oxygen atoms are indicated in red and nitrogen atoms in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 16

Electrostatic potential surfacesof theparacellular scaffolds. (A) Cldn4 Pore I, (B) Cldn5 Pore I, (C) Cldn4 Pore II and (D) Cldn5 Pore II are represented. Electrostatic potential surfaces were computed with the Adaptive Poisson-Boltzmann Solver software . Isosurfaces are drawn with a red-white-blue color scale ranging from −5.0 (red) to + 5.0 (blue) kT/e. (A,C) Ref. . Copyright 2022 by John Wiley & Sons, Inc. Reproduced with permission of John Wiley & Sons, Inc. in the format of Journal/magazine via Copyright Clearance Center. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)