988 research outputs found
Targeting Tight Junctions in Nanomedicine: a Molecular Modeling Perspective
No full textMolecular Dynamics Simulations of Claudin Paracellular Channel
Structural Mechanism of ω-Currents in a Mutated Kv7.2 Voltage Sensor Domain from Molecular Dynamics Simulations
No full textActivation of voltage-gated ion channels is regulated by conformational changes of the voltage sensor domains (VSDs), four water- and ion-impermeable modules peripheral to the central, permeable pore domain. Anomalous currents, defined as ω-currents, have been recorded in response to mutations of residues on the VSD S4 helix and associated with ion fluxes through the VSDs. In humans, gene defects in the potassium channel Kv7.2 result in a broad range of epileptic disorders, from benign neonatal seizures to severe epileptic encephalopathies. Experimental evidence suggests that the R207Q mutation in S4, associated with peripheral nerve hyperexcitability, induces ω-currents at depolarized potentials, but the fine structural details are still elusive. In this work, we use atom-detailed molecular dynamics simulations and a refined model structure of the Kv7.2 VSD in the active conformation in a membrane/water environment to study the effect of R207Q and four additional mutations of proven clinical importance. Our results demonstrate that the R207Q mutant shows the most pronounced increase of hydration in the internal VSD cavity, a feature favoring the occurrence of ω-currents. Free energy and kinetics calculations of sodium permeation through the native and mutated VSD indicate as more favorable the formation of a cationic current in the latter. Overall, our simulations establish a mechanistic linkage between genetic variations and their physiological outcome, by providing a computational description that includes both thermodynamic and kinetic features of ion permeation associated with ω-currents
Computational study of ion permeation through claudin‐4 paracellular channels
No full textClaudins (Cldns) form a large family of protein homologs that are essential for the assembly of paracellular tight junctions (TJs), where they form channels or barriers with tissue-specific selectivity for permeants. In contrast to several family members whose physiological role has been identified, the function of claudin 4 (Cldn4) remains elusive, despite experimental evidence suggesting that it can form anion-selective TJ channels in the renal epithelium. Computational approaches have recently been employed to elucidate the molecular basis of Cldns’ function, and hence could help in clarifying the role of Cldn4. In this work, we use structural modeling and all-atom molecular dynamics simulations to transfer two previously introduced structural models of Cldn-based paracellular complexes to Cldn4 to reproduce a paracellular anion channel. Free energy calculations for ionic transport through the pores allow us to establish the thermodynamic properties driving the ion-selectivity of the structures. While one model shows a cavity permeable to chloride and repulsive to cations, the other forms barrier to the passage of all the major physiological ions. Furthermore, our results confirm the charge selectivity role of the residue Lys65 in the first extracellular loop of the protein, rationalizing Cldn4 control of paracellular permeability
Multiscale modelling of claudin-based assemblies: a magnifying glass for novel structures of biological interfaces
No full textClaudins (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
The impact of pathogenic and artificial mutations on Claudin-5 selectivity from molecular dynamics simulations
No full textTight-junctions (TJs) are multi-protein complexes between adjacent endothelial or epithelial cells. In the blood-brain-barrier (BBB), they seal the paracellular space and the Claudin-5 (Cldn5) protein forms their backbone. Despite the fundamental role in brain homeostasis, little is known on Cldn5-based TJ assemblies. Different structural models were suggested, with Cldn5 protomers generating paracellular pores that restrict the passage of ions and small molecules. Recently, the first Cldn5 pathogenic mutation, G60R, was identified and shown to induce Cl−-selective channels and Na+ barriers in BBB TJs, providing an excellent opportunity to validate the structural models. Here, we used molecular dynamics to study the permeation of ions and water through two distinct G60R-Cldn5 paracellular architectures. Only the so-called Pore I reproduces the functional modification observed in experiments, displaying a free energy (FE) minimum for Cl− and a barrier for Na+ consistent with anionic selectivity. We also studied the artificial Q57D and Q63D mutations in the constriction region, Q57 being conserved in Cldns except for cation permeable homologs. In both cases, we obtain FE profiles consistent with facilitated passage of cations. Our calculations provide the first in-silico description of a Cldn5 pathogenic mutation, further assessing the TJ Pore I model and yielding new insight on BBB’s paracellular selectivity
Computational Assessment of Different Structural Models for Claudin-5 Complexes in Blood-Brain Barrier Tight Junctions
No full textThe blood-brain barrier (BBB) strictly regulates the exchange of ions and molecules between the blood and the central nervous system. Tight junctions (TJs) are multimeric structures that control the transport through the paracellular spaces between the adjacent brain endothelial cells of the BBB. Claudin-5 (Cldn5) proteins are essential for TJ formation and assemble into multiprotein complexes via cis-interactions within the same cell membrane and trans-interactions across two contiguous cells. Despite the relevant biological function of Cldn5 proteins and their role as targets of brain drug delivery strategies, the molecular details of their assembly within TJs are still unclear. Two different structural models have been recently introduced, in which Cldn5 dimers belonging to opposite cells join to generate paracellular pores. However, a comparison of these models in terms of ionic transport features is still lacking. In this work, we used molecular dynamics simulations and free energy (FE) calculations to assess the two Cldn5 pore models and investigate the thermodynamic properties of water and physiological ions permeating through them. Despite different FE profiles, both structures present single/multiple FE barriers to ionic permeation, while being permissive to water flux. These results reveal that both models are compatible with the physiological role of Cldn5 TJ strands. By identifying the protein-protein surface at the core of TJ Cldn5 assemblies, our computational investigation provides a basis for the rational design of synthetic peptides and other molecules capable of opening paracellular pores in the BBB
Ion and water permeation through claudin-10b and claudin-15 paracellular channels
No full textThe structural scaffold of epithelial and endothelial tight junctions (TJs) comprises multimeric strands of claudin (Cldn) proteins that anchor adjacent cells and control the paracellular flux of water and solutes. Based on the permeability properties they confer to the TJs, Cldns are classified as channel- or barrier-forming. For instance, Cldn10b, expressed in kidneys, lungs, and other tissues, displays high permeability for cations and low permeability for water. Along with its high sequence similarity to the cation- and water-permeable TJ protein Cldn15, this makes Cldn10b a valuable test case for investigating the molecular determinants of paracellular transport. In lack of high-resolution experimental information on TJ architectures, here we use molecular dynamics simulations to determine whether atomistic models recapitulate the differences in ion and water transport between of Cldn10b and Cldn15. Our data, based on extensive standard simulations and free energy calculations, reveal that Cldn10b models form cation-permeable pores narrower than Cldn15, which, together with the stable coordination of Na+ ions to acidic pore-lining residues (E153, D36, D56), limit the passage of water molecules. By providing a mechanism driving a peculiar case of paracellular transport, these results provide a structural basis for the specific permeability properties of Cldn subtypes that define their physiological role
A multi-pore model of the blood–brain barrier tight junction strands recapitulates the permeability features of wild-type and mutant claudin-5
No full textIn the blood–brain barrier (BBB), endothelial cells are joined by tight junctions (TJs), multi-protein assemblies that seal the paracellular space and restrict molecular transport. Among the BBB TJ proteins, Claudin-5 (Cldn15) is the most abundant one. Structural models for claudin complexes, first introduced for channel-forming, selectively permeable claudins, comprise protomers arranged to form paracellular pores that regulate transport by electrostatic and/or steric effects arising from pore-lining residues. With limited exceptions, computational studies explored oligomers of only a few subunits, while TJs are formed by extended polymeric strands. Here, we employ multi-microsecond all-atom molecular dynamics and free-energy (FE) calculations to study two distinct models of TJ-forming Cldn15 complexes, called multi-Pore I and multi-Pore II, each comprising 16 protomers arranged around three adjacent pores. FE calculations of water and ions permeation reveal that, in both models, ion transport is hindered by FE barriers higher than in single pores. Moreover, only the multi-Pore I model captures the Cldn15 G60R variant's effect, making it anion-permeable. The results provide insights into Cldn15 structure and function and validate a structural model of BBB TJs useful for studying barrier impairment in brain diseases and for developing therapeutic approaches
Fine morphology of the myrmecophilous larva of Paussus kannegieteri (Coleoptera: Carabidae: Paussinae: Paussini). Corresponding author
No full textFIGURES 13–18. Paussus kannegieteri third instar larva: 13, thorax, left lateral view; 14, thorax, dorsal view; 15, mesothoracic spiracle; 16, metathoracic spiracle-like structure; 17, mesothoracic leg, anterolateral view; 18, apex of metathoracic leg with lanceolate setae, posterolateral view. CO = coxa, ls = lanceolate setae, m = membrane, ME = mesonotum, MT = metanotum, pe = peritreme, PR = pronotum, un = claw. Scale bars: Figs. 13–14 = 500 µm; Fig. 15 = 10 µm; Fig. 16 = 20 µm; Fig. 17 = 200 µm; Fig. 18 = 50 µm.Published as part of Giulio, Andrea Di, 2008, Fine morphology of the myrmecophilous larva of Paussus kannegieteri (Coleoptera: Carabidae: Paussinae: Paussini), pp. 37-50 in Zootaxa 1741 on page 44, DOI: 10.5281/zenodo.18152
A refined model of claudin-15 tight junction paracellular architecture by molecular dynamics simulations.
No full textTight-junctions between epithelial cells of biological barriers are specialized molecular structures that regulate the flux of solutes across the barrier, parallel to cell walls. The tight-junction backbone is made of strands of transmembrane proteins from the claudin family, but the molecular mechanism of its function is still not completely understood. Recently, the crystal structure of a mammalian claudin-15 was reported, displaying for the first time the detailed features of transmembrane and extracellular domains. Successively, a structural model of claudin-15-based paracellular channels has been proposed, suggesting a putative assembly that illustrates how claudins associate in the same cell (via cis interactions) and across adjacent cells (via trans interactions). Although very promising, the model offers only a static conformation, with residues missing in the most important extracellular regions and potential steric clashes. Here we present detailed atomic models of paracellular single and double pore architectures, obtained from the putative assembly and refined via structural modeling and all-atom molecular dynamics simulations in double membrane bilayer and water environment. Our results show an overall stable configuration of the complex with a fluctuating pore size. Extracellular residue loops in trans interaction are able to form stable contacts and regulate the size of the pore, which displays a stationary radius of 2.5-3.0 Å at the narrowest region. The side-by-side interactions of the cis configuration are preserved via stable hydrogen bonds, already predicted by cysteine crosslinking experiments. Overall, this work introduces an improved version of the claudin-15-based paracellular channel model that strengthens its validity and that can be used in further computational studies to understand the structural features of tight-junctions regulation
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