Jonas Protze

Mechanism of Clostridium perfringens Enterotoxin Interaction with Claudin-3/-4 Protein Suggests Structural Modifications of the Toxin to Target Specific Claudins

Background: Clostridium perfringens enterotoxin (CPE) binds to a subset of claudin tight junction proteins. Results: The molecular interface of the CPE-claudin interaction was mapped. Conclusion: Claudin-3 and -4 interact with CPE in the same orientation but in different modes. Significance: The mechanistic insights might advance design of CPE-based claudin modulators to improve paracellular drug delivery or to target claudin-overexpressing tumors. Claudins (Cld) are essential constituents of tight junctions. Domain I of Clostridium perfringens enterotoxin (cCPE) binds to the second extracellular loop (ECL2) of a subset of claudins, e.g. Cld3/4 and influences tight junction formation. We aimed to identify interacting interfaces and to alter claudin specificity of cCPE. Mutagenesis, binding assays, and molecular modeling were performed. Mutation-guided ECL2 docking of Cld3/4 onto the crystal structure of cCPE revealed a common orientation of the proposed ECL2 helix-turn-helix motif in the binding cavity of cCPE: residues Leu150/Leu151 of Cld3/4 bind similarly to a hydrophobic pit formed by Tyr306, Tyr310, and Tyr312 of cCPE, and Pro152/Ala153 of Cld3/4 is proposed to bind to a second pit close to Leu223, Leu254, and Leu315. However, sequence variation in ECL2 of these claudins is likely responsible for slightly different conformation in the turn region, which is in line with different cCPE interaction modes of Cld3 and Cld4. Substitutions of other so far not characterized cCPE residues lining the pocket revealed two spatially separated groups of residues (Leu223, Asp225, and Arg227 and Leu254, lle258, and Asp284), which are involved in binding to Cld3 and Cld4, albeit differently. Involvement of Asn148 of Cld3 in cCPE binding was confirmed, whereas no evidence for involvement of Lys156 or Arg157 was found. We show structure-based alteration of cCPE generating claudin binders, which interact subtype-specific preferentially either with Cld3 or with Cld4. The obtained mutants and mechanistic insights will advance the design of cCPE-based modulators to target specific claudin subtypes related either to paracellular barriers that impede drug delivery or to tumors.

On the Interaction of Clostridium perfringens Enterotoxin with Claudins

Clostridium perfringens causes one of the most common foodborne illnesses, which is largely mediated by the Clostridium perfringens enterotoxin (CPE). The toxin consists of two functional domains. The N-terminal region mediates the cytotoxic effect through pore formation in the plasma membrane of the mammalian host cell. The C-terminal region (cCPE) binds to the second extracellular loop of a subset of claudins. Claudin-3 and claudin-4 have been shown to be receptors for CPE with very high affinity. The toxin binds with weak affinity to claudin-1 and -2 but contribution of these weak binding claudins to CPE-mediated disease is questionable. cCPE is not cytotoxic, however, it is a potent modulator of tight junctions. This review describes recent progress in the molecular characterization of the cCPE-claudin interaction using mutagenesis, in vitro binding assays and permeation studies. The results promote the development of recombinant cCPE-proteins and CPE-based peptidomimetics to modulate tight junctions for improved drug delivery or to treat tumors overexpressing claudins.

Claudin-3 and Claudin-5 Protein Folding and Assembly into the Tight Junction Are Controlled by Non-conserved Residues in the Transmembrane 3 (TM3) and Extracellular Loop 2 (ECL2) Segments

Background: The transmembrane claudins assemble into polymeric tight junction strands. Results: Residues involved in differential folding and assembly of claudin-3 and claudin-5 were identified. Conclusion: Subtype-specific cis-dimerization contributes to the differing ultrastructure of tight junction strands. Significance: The molecular insights improve the understanding of the formation of paracellular barriers to molecules. The mechanism of tight junction (TJ) assembly and the structure of claudins (Cldn) that form the TJ strands are unclear. This limits the molecular understanding of paracellular barriers and strategies for drug delivery across tissue barriers. Cldn3 and Cldn5 are both common in the blood-brain barrier but form TJ strands with different ultrastructures. To identify the molecular determinants of folding and assembly of these classic claudins, Cldn3/Cldn5 chimeric mutants were generated and analyzed by cellular reconstitution of TJ strands, live cell confocal imaging, and freeze-fracture electron microscopy. A comprehensive screening was performed on the basis of the rescue of mutants deficient for strand formation. Cldn3/Cldn5 residues in transmembrane segment 3, TM3 (Ala-127/Cys-128, Ser-136/Cys-137, Ser-138/Phe-139), and the transition of TM3 to extracellular loop 2, ECL2 (Thr-141/Ile-142) and ECL2 (Asn-148/Asp-149, Leu-150/Thr-151, Arg-157/Tyr-158), were identified to be involved in claudin folding and/or assembly. Blue native PAGE and FRET assays revealed 1% n-dodecyl β-d-maltoside-resistant cis-dimerization for Cldn5 but not for Cldn3. This homophilic interaction was found to be stabilized by residues in TM3. The resulting subtype-specific cis-dimer is suggested to be a subunit of polymeric TJ strands and contributes to the specific ultrastructure of the TJ detected by freeze-fracture electron microscopy. In particular, the Cldn5-like exoplasmic face-associated and particle-type strands were found to be related to cis-dimerization. These results provide new insight into the mechanisms of paracellular barrier formation by demonstrating that defined non-conserved residues in TM3 and ECL2 of classic claudins contribute to the formation of TJ strands with differing ultrastructures.

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

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. 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. 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.

Targeting and alteration of tight junctions by bacteria and their virulence factors such as Clostridium perfringens enterotoxin

The integrity of tight junctions, which regulate paracellular permeability, is challenged by many bacterial pathogens. This is caused by inflammatory responses triggered by pathogens and direct interaction of bacteria or their toxins with host epithelial cells. In some cases, tight junction proteins represent receptors for cell surface proteins or toxins of the pathogen, such as Clostridium perfringens enterotoxin (CPE). CPE causes diarrhea and cramps—the symptoms of a common foodborne illness, caused by C. perfringens type A. It uses a subgroup of the claudin family of tight junction proteins as receptors and forms pores in the membrane of intestinal epithelial cells. Ca2+ influx through these pores finally triggers cell damage. In this review, we summarize tight junction targeting and alteration by a multitude of different microorganisms such as C. perfringens, Escherichia coli, Helicobacter pylori, Salmonella typhimurium, Shigella flexneri, Vibrio cholerae, Yersinia enterocolitica, protozoan parasites, and their proteins. A focus is drawn towards CPE, the interaction with its receptors, cellular, and pathophysiological consequences for the intestinal epithelium. In addition, we portend to the use of CPE-based claudin modulators for drug delivery as well as diagnosis and therapy of cancer.

Molecular and structural transmembrane determinants critical for embedding claudin-5 into tight junctions reveal a distinct four-helix bundle arrangement

The mechanism of TJ (tight junction) assembly and the structure of TJ strand-forming Cldns (claudins) are unclear. To identify determinants of assembly of blood-brain barrier-related Cldn3 and Cldn5, chimaeric mutants were analysed by cellular reconstitution of TJ strands and live-cell imaging. On the basis of the rescue of mutants deficient for strand formation, we identified Cldn5 residues (Cys128, Ala132, Ile142, Ala163, Ile166 and Leu174) involved in Cldn folding and assembly. Experimental results were combined with structural bioinformatics approaches. Initially the experimentally validated previous model of the ECL2 (extracellular loop 2) of Cldn5 was extended to the flanking transmembrane segments (TM3/TM4). A coiled-coil interface probably caused by alternating small and large residues is supported by concomitant knob-into-hole interactions including Cldn5-specific residues identified in the present paper. To address arrangement of the TMs in a four-helix bundle, data from evolutionary sequence couplings and comparative modelling of intramolecular interfaces in the transmembrane region of Cldns led to a complete Cldn5 model. Our suggested Cldn subtype-specific intramolecular interfaces that are formed by conserved coiled-coil motifs and non-conserved residues in distinct TM positions were confirmed by the recently released crystal structure of Cldn15. The identified molecular and structural determinants essentially contribute to assembly of Cldns into TJ strands.

Polar and charged extracellular residues conserved among barrier-forming claudins contribute to tight junction strand formation

Claudins (Cldn) form the backbone of tight junction (TJ) strands and thereby regulate paracellular permeability for solutes and water. Polymeric strands are formed by homo‐ and heterophilic cis‐ and trans‐interactions between claudin protomers. Crystal structures of some claudins have been resolved; however, the mechanism by which claudins assemble into TJ strands remains unclear. To elucidate strand architecture, TJ‐like strands were reconstituted in HEK293 cells by claudin transfection. Determinants of prototypic, classic barrier‐forming claudins (Cldn1, ‐3, and ‐5) involved in strand formation were analyzed by mutagenesis. The capability of claudin constructs to interact in trans and to form strands was investigated by cell contact–enrichment assays and freeze‐fracture electron microscopy. Residues in extracellular loops 1 and 2 of the claudins affecting strand formation were identified. Using homology modeling and molecular docking, we tested working concepts for the arrangement of claudin protomers within TJ strands. We show that the charge of Lys65 in Cldn1 and Glu158 in Cldn3, but not of Arg30 or Asp145 in Cldn3, and the polarity of Gln56 and Gln62 in Cldn3 and of Gln57 in Cldn5 are necessary for TJ strand formation. These residues are all conserved among barrier‐forming classic claudins. The results contribute to mechanistic understanding of claudin‐based regulation of paracellular permeability.

Determinants contributing to claudin ion channel formation

Pore‐forming properties of claudins (Cld) are likely defined by residues of their first extracellular loop (ECL1). Detailed mechanisms are unclear. MDCK cells overexpressing FLAG‐Cld‐1 wild‐type and mutants were characterized by transepithelial resistance (TER) and ion permeability measurements. Replacing ECL1 residues of sealing Cld‐1 by corresponding Cld‐2 residues we aimed to identify new determinants responsible for sealing and/or pore formation. We found that E48K and S53E substitutions in human Cld‐1 strongly reduced TER and increased permeability for Na+ and Cl−. In contrast, K65D, D68S, and other single substitutions showed no significant change of TER and permeability for Na+ and Cl−. Double substitution S53E/K65D did not change TER and ion permeability, whereas S53E/D68S decreased TER, albeit weaker than S53E. Ratio of permeabilities for Na+ and Cl− revealed no clear charge specificity of the pore induced by S53E or S53E/D68S in Cld‐1, suggesting that primarily S53 and potentially D68 in Cld‐1 are involved in sealing of the paracellular cleft and that charge‐unselective pores may be induced by substituting S53E.

Membrane-traversing mechanism of thyroid hormone transport by monocarboxylate transporter 8

Monocarboxylate transporter 8 (MCT8) mediates thyroid hormone (TH) transport across the plasma membrane in many cell types. In order to better understand its mechanism, we have generated three new MCT8 homology models based on sugar transporters XylE in the intracellular opened (PDB ID: 4aj4) and the extracellular partly occluded (PDB ID: 4gby) conformations as well as FucP (PDB ID: 3o7q) and GLUT3 (PDB ID: 4zwc) in the fully extracellular opened conformation. T3-docking studies from both sides revealed interactions with His192, His415, Arg445 and Asp498 as previously identified. Selected mutations revealed further transport-sensitive positions mainly at the discontinuous transmembrane helices TMH7 and 10. Lys418 is potentially involved in neutralising the charge of the TH substrate because it can be replaced by charged, but not by uncharged, amino acids. The side chain of Thr503 was hypothesised to stabilise a helix break at TMH10 that undergoes a prominent local shift during the transport cycle. A T503V mutation accordingly affected transport. The aromatic Tyr419, the polar Ser313 and Ser314 as well as the charged Glu422 and Glu423 lining the transport channel have been studied. Based on related sugar transporters, we suggest an alternating access mechanism for MCT8 involving a series of amino acid positions previously and newly identified as critical for transport.

Defining a Conformational Consensus Motif in Cotransin-Sensitive Signal Sequences: A Proteomic and Site-Directed Mutagenesis Study

The cyclodepsipeptide cotransin was described to inhibit the biosynthesis of a small subset of proteins by a signal sequence-discriminatory mechanism at the Sec61 protein-conducting channel. However, it was not clear how selective cotransin is, i.e. how many proteins are sensitive. Moreover, a consensus motif in signal sequences mediating cotransin sensitivity has yet not been described. To address these questions, we performed a proteomic study using cotransin-treated human hepatocellular carcinoma cells and the stable isotope labelling by amino acids in cell culture technique in combination with quantitative mass spectrometry. We used a saturating concentration of cotransin (30 micromolar) to identify also less-sensitive proteins and to discriminate the latter from completely resistant proteins. We found that the biosynthesis of almost all secreted proteins was cotransin-sensitive under these conditions. In contrast, biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Cotransin sensitivity of signal sequences was neither related to their length nor to their hydrophobicity. Instead, in the case of signal anchor sequences, we identified for the first time a conformational consensus motif mediating cotransin sensitivity.