Biology subjects

Maragliano, L.

Publications and source records attributed to Maragliano, L..

13 recordsLinked to original sources

bioRxiv2026

Controlled Blood-Brain Barrier Modulation by a High-Affinity Claudin-5 Peptide Binder

The blood-brain barrier (BBB) is a specialized interface that tightly regulates the exchange of molecules between the bloodstream and the brain. Its barrier function relies on a monolayer of brain endothelial cells sealed by tight junctions (TJs) that restrict paracellular flux through claudin-5 (CLDN5) multimeric complexes. To improve the delivery of nutrients and drugs to the brain, CLDN5-competitive peptides are promising carriers for creating size-controlled, temporary openings in the paracellular pathway. Here, we combine generative protein design and atomistic simulations to design ST9, a peptide with high nanomolar affinity for CLDN5. Compared with f1-C5C2, a CLDN5-binding peptide that we previously reported, ST9 induces a rapid, transient, size-controlled, and fully reversible increase in paracellular permeability, without altering CLDN5 expression or the proteomic profile of brain endothelial cells, indicating distinct mechanisms of TJ destabilization. This work provides a promising approach for developing next-generation BBB-opening agents to effectively treat neurological diseases.

neuroscience↗

bioRxiv2026

Molecular mechanism of partially open Kv7.2 gate stabilization by gain-of-function variants

Gain-of-function (GoF) variants in the Kv7.2 channel are associated with a clinically relevant subset of neurodevelopmental disorders. While most GoF substitutions identified so far affect the voltage-sensing domain, we recently described three mutations in the intracellular-facing activation gate (AG), G313S, A317T, and L318V. Electrophysiological recordings showed that these variants increase macroscopic current density and enhance channel open probability. Consistently, molecular dynamics (MD) simulations revealed that they hinder complete channel closure by destabilizing the closed AG and increasing hydration of the central cavity (CC). Whether this partially open conformation can support K+ permeation, however, remained an open question. Here, we combined long-timescale atomistic simulations and simulations with applied electric fields to evaluate the stability of these mutant-associated AG states over longer timescales and their functional relevance. In new trajectories, all three substitutions consistently shifted the closed intracellular gate toward a widened, water-accessible conformation, accompanied by increased CC hydration. We then assessed the functional significance of this partially open state by simulating the A317T channel under applied electric fields. The conformation supported K+ translocation, whereas the closed WT pore remained impermeable under all tested voltages. When simulations were started from open channel conformations, both WT and A317T conducted K+ ions, indicating that the main effect of the substitution is to destabilize closure of the intracellular gate rather than to alter the fully conductive open state. Together, these data show that AG GoF variants can generate an intermediate gate conformation that permits ion permeation, providing a mechanistic link between mutant-induced pore remodeling and Kv7.2 dysfunction in KCNQ2-related disease.

biophysics↗

bioRxiv2025

A multi-pore model of the blood-brain barrier tight junction strands recapitulates the permeability features of wild-type and mutant claudin-5

In the blood-brain barrier (BBB), endothelial cells are joined together by multi-protein assemblies called tight junctions (TJs), which seal the paracellular space and restrict the passage of substances. Among the proteins forming BBB TJs, Claudin-5 (Cldn5) is the most abundant one. Structural models for complexes of Cldn5 and other claudins have been proposed and assessed via experimental and computational approaches. In these models, first introduced for channel-forming, selectively permeable claudins, protomers are arranged to form pores in the paracellular space that regulate transport by electrostatic and/or steric effects arising from the pore-lining residues. With limited exceptions, however, previous computational studies focused on oligomers of only a few subunits, while extended polymeric claudin strands form TJs. Here, we employ multi-microsecond all-atom molecular dynamics and free energy (FE) calculations to study two distinct models of TJ-forming Cldn5 complexes, called multi-Pore I and multi-Pore II, each comprising sixteen protomers arranged around three adjacent pores. Free energy calculations of water and ions permeation across the pores reveal that, in both models, the passage of ions is hindered by FE barriers, which are higher than in single-pore architectures. Moreover, only the multi-Pore I structural model recapitulates the effect of the G60R variant of Cldn5, making it permeable to anions. The results provide new insights into Cldn5 structure and function and validate a structural model of BBB TJs that may be useful for the study of barrier impairment in brain diseases and for developing new therapeutic approaches.

neuroscience↗

bioRxiv2024

Ion and water permeation through Claudin-10b paracellular channels

The structural scaffold of epithelial and endothelial tight junctions (TJ) comprises multimeric strands of claudin (Cldn) proteins, which 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. Some of them, however, show mixed features. 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 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 study two atomistic models of Cldn10b strands and compare their ion and water transport with those of Cldn15. Our data, based on extensive standard simulations and Free Energy calculations, reveal that both 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 isoforms that define their physiological role. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/601692v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1ffef0aorg.highwire.dtl.DTLVardef@1a67a8corg.highwire.dtl.DTLVardef@b7a31aorg.highwire.dtl.DTLVardef@35295c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

CONSTITUTIVE OPENING OF THE Kv7.2 PORE ACTIVATION GATE CAUSES KCNQ2-DEVELOPMENTAL ENCEPHALOPATHY

Pathogenic variants in KCNQ2 encoding for Kv7.2 voltage-gated potassium channel subunits cause developmental encephalopathies (KCNQ2-encephalopathies), both with and without epilepsy. We herein describe the clinical, in vitro and in silico features of two encephalopathy-causing variants (A317T, L318V) in Kv7.2 affecting two consecutive residues in the S6 activation gate undergoing large structural rearrangements during pore opening. Currents through these mutant channels displayed increased density, hyperpolarizing shifts in activation gating, and insensitivity to phosphatidylinositol 4,5-bisphosphate (PIP2), a critical regulator of Kv7 channel function; all these features are consistent with a strong gain-of-function effect. An increase in single-channel open probability, with no change in membrane abundance or single-channel conductance, was responsible for the observed gain-of-function effects. All-atoms Molecular Dynamics simulations revealed that the mutations widened the inner pore gate and stabilized a constitutively open channel configuration in the closed state, with minimal effects on the open conformation. Thus, a PIP2-independent stabilization of the inner pore gate open configuration is a novel molecular pathogenetic mechanism for KCNQ2-developmental encephalopathies.

biophysics↗

bioRxiv2024

A claudin5-binding peptide enhances the permeability of the blood-brain-barrier

The blood-brain barrier (BBB) is essential to maintain brain homeostasis and healthy conditions but it also prevents drugs from reaching brain cells. In the BBB, tight junctions (TJs) are multi-protein complexes located at the interface between adjacent brain endothelial cells that regulate paracellular diffusion and claudin-5 (CLDN5) is the major component of the TJ portfolio, playing a pivotal role in restricting the paracellular traffic. In view of obtaining fine control over the transport across the BBB, the use of competing peptides able to bind CLDN5 to induce transient and regulated permeabilization of the paracellular passage is emerging as a potentially translatable strategy for clinical applications. In this work, we designed and tested short peptides with improved solubility and biocompatibility using a combined approach that involved structural modeling techniques and in vitro validation, generating a robust workflow for the design, screening, and optimization of peptides for the modulation of the BBB paracellular permeability. We designed a selection of 11- to 16-mer compounds derived from the first CLDN5 extracellular domain and from the CLDN5-binding domain of Clostridium perfringens enterotoxin and determined their efficiency in enhancing BBB permeability. The computational analysis classified all tested peptides based on solubility and affinity to CLDN5, and provided atom-level details of the binding process. From our screening, we identified a novel CLDN5-derived peptide, here called f1-C5C2, which demonstrated good solubility in biological media, efficient binding to CLDN5 subunits, and capability to increase permeability at low concentrations. The peptidomimetic in silico/in vitro strategy described here can achieve a transient and reversible permeabilization of the BBB with potential applications in the pharmacological treatment of brain diseases. HIGHLIGHTSO_LIWater-soluble peptidomimetics are used to competitively bind claudin-5 tight junction proteins and increase the permeability of the blood-brain barrier; C_LIO_LITrans-endothelial electrical resistance and dissociation constant measurements demonstrate the binding affinity of the peptide f1-C5C2 for claudin-5; C_LIO_LIUnbinding free energy calculations correlated with experimental results and provided information on the protein-peptide binding interface. C_LIO_LIIncubation with the peptide f1-C5C2 allows paracellular transport of 4K, but not 70K, dextran. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/591687v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@70fe54org.highwire.dtl.DTLVardef@496a28org.highwire.dtl.DTLVardef@1d89a81org.highwire.dtl.DTLVardef@187acec_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

The Impact of Pathogenic and Artificial Mutations on Claudin-5 Selectivity from Molecular Dynamics Simulations

Tight junctions (TJs) are multi-protein complexes at the interface between adjacent endothelial or epithelial cells. In the blood-brain barrier (BBB), they are responsible for sealing the paracellular spaces and their backbone is formed by Claudin-5 (Cldn5) proteins. Despite the important role in preserving brain homeostasis, little is known on how Cldn5 oligomers assemble. Different structural models have been suggested, where Cldn5 protomers from opposite cells associate to generate paracellular pores that do not allow the passage of ions or small molecules. Recently, the first Cldn5 pathogenic mutation, G60R, was identified and shown to induce anion selectivity in the BBB TJs. This offers an excellent opportunity to further assess the structural models. In this work, we performed umbrella sampling molecular dynamics simulations to study the permeation of single Na+, Cl- and H2O through two distinct G60R Cldn5 paracellular models. Only one of them, called Pore I, reproduces the functional modification observed in the experiments, displaying a free energy (FE) minimum for Cl- and a barrier for Na+ at the central constriction, consistent with the formation of an anionic channel. To further test the validity of the model, we performed the same calculations for the Q57D and the Q63D mutants, which affect two side-chains in the constriction site. In particular, Q57 is conserved among various Cldns, with few exceptions such as the two cation permeable homologs Cldn15 and Cldn10b. In both cases, we obtain that the FE profiles are modified with respect to the wild-type system, facilitating the passage of cations. Our calculations are the first in-silico description of the effect of a Cldn5 pathogenic mutation, and provide a further assessment of the Pore I model for Cldn5-based TJ architectures, yielding new atom-detailed insight on the selective permeability of the paracellular spaces in BBB.

neuroscience↗

bioRxiv2022

Molecular Dynamics Simulations of Ion Permeation in human NaV Channels

The recent determination of cryo-EM structures of voltage-gated sodium (Nav) channels has revealed many details of these proteins. However, knowledge of ionic permeation through the Nav pore remains limited. In this work, we performed atomistic molecular dynamics simulations to study the structural features of various neuronal Nav channels based on homology modeling of the cryo-EM structure of the human Nav1.4 channel and, in addition, on the more recent resolved configuration for Nav1.2. In particular, single Na+ permeation events during standard MD runs suggest that the ion resides in the inner part of the Nav selectivity filter (SF). On-the-fly free-energy parametrization (OTFP) temperature accelerated molecular dynamics (TAMD) was also used to calculate two-dimensional free energy surfaces (FESs) related to single/double Na+ translocation through the SF of the Nav1.2 homology model. The same thermodynamic analysis was performed on the cryo-EM based Nav1.2 configuration. These additional simulations revealed distinct mechanisms for single and double Na+ permeation through the wild-type SF, which has a charged lysine in the DEKA ring. In particular, the extracted protein-ion configurations are not accessible by the other modified SFs tested by TAMD/OTFP. Overall, the description of these mechanisms gives us new insights into ion conduction in human Nav cryo-EM based configurations, that could advance understanding of these systems and how they differ from potassium and bacterial Nav channels. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/509656v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@53d852org.highwire.dtl.DTLVardef@9f17aaorg.highwire.dtl.DTLVardef@cacc7borg.highwire.dtl.DTLVardef@36bd64_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

In silico conformational features of botulinum toxins A1 and E1 according to the intraluminal acidification

Although the botulinum neurotoxins (BoNTs) are among the most toxic compounds found in nature, their molecular mechanism of action is far from being elucidated. A key event is the conformational transition due to the acidification of the interior of synaptic vesicles, and leading to the translocation of the BoNT catalytic domain into the neuronal cytosol. To investigate these conformational variations, homology modelling and atomistic simulations are combined to explore the internal dynamics of the subtypes BoNT/A1, the most-used in medical applications, and BoNT/E1, the most kinetically efficient. This first simulation study of di-chain BoNTs in closed and open states includes the effects of neutral and acidic pH. The conformational mobility is driven by domains displacements; the ganglioside binding site in the receptor binding domain, the translocation domain (HCNT) switch and the belt helix visit multiple conformations depending on the primary sequence and on the pH. Fluctuations of the belt helix are observed for closed conformations of the toxins and at acidic pH, and patches of more accessible residues appear in the same conditions in the core translocation domain HCNT. These findings suggest that during translocation, the larger mobility of belt could be transmitted to HCNT, leading to a favorable interaction of HCNT residues with the non-polar membrane environment. Key ContributionThe molecular dynamics simulations presented here provide a structural and functional annotation of full-length BoNTs composed by two distinct protein chains. Two different conformations (open and closed) as well as two different protonation states, corresponding to acidic and neutral pH, have been considered. Results from the present work supports a model of mobility in which the individual domains fluctuate around stable conformations and the overall structure mobility arise from relative displacements of the domains.

neuroscience↗

bioRxiv2022

Computational Study of Ion Permeation Through Claudin-4 Paracellular Channels

Claudins (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 Cldn4 role. 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, in order to reproduce a paracellular anion channel. Free energy (FE) 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.

biophysics↗

bioRxiv2022

Computational Assessment of Different Structural Models for Claudin-5 Complexes in Blood-Brain-Barrier Tight-Junctions

The 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 adjacent brain endothelial cells of the BBB. Claudin-5 (Cldn5) proteins are essential for the TJ formation and assemble into multi-protein 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.

neuroscience↗

bioRxiv2021

Free energy and kinetics of cAMP permeation through connexin26 hemichannel with and without voltage

The connexin family is a diverse group of highly regulated non-{beta}-barrel wide-pore channels permeable to biological signaling molecules. Despite their critical roles in mediating selective molecular signaling in health and disease, the molecular basis of permeation through these pores remains unclear. Here, we report the thermodynamics and kinetics of binding and transport of a second messenger, adenosine-3,5-cyclophosphate (cAMP), through a connexin26 hemichannel. Inward and outward fluxes of cAMP were first obtained from 4 s simulations with voltages and multiple cAMPs in solution. The results are compared with the intrinsic potential of mean force (PMF) and the mean first passage times (MFPTs) of a single cAMP in the absence of voltage, obtained from a total of 16.5 s of multi-replica Voronoi-tessellated Markovian milestoning simulations. The computed transit times through the pore correspond well to existing experimental data. Both voltage simulations and milestoning simulations revealed two cAMP binding sites with binding constants and dissociation rates computed from PMF and MFPTs. The protein dipole inside the pore produces an asymmetric PMF, reflected in unequal cAMP MFPTs in each direction once within the pore. The free energy profiles under voltages derived from intrinsic PMF provided a unified understanding of directional transition rates with/without voltage, and revealed the unique role of channel polarity and the mobile electrolyte within a wide pore on the total free energy. In addition, we show how these factors influence the cAMP dipole vector during permeation, and how cAMP affects the local and non-local pore diameter in a position-dependent manner. Significance StatementConnexins are wide-pore channels permeable to cellular signaling molecules. They mediate molecular signaling crucial in physiology, pathology, and development; mutations in connexins cause human pathologies. However, the fundamental structural, thermodynamic, and kinetic determinants of molecular permeability properties are unknown. Using multiple molecular dynamics simulation techniques, we report, for the first time, an in-depth investigation of the free energy and the directional transition rates of an important biological signaling molecule, cAMP, through a connexin channel. We reveal the energetics and binding sites that determine the cAMP flux, and the effects of mobile ions and external electrical field on the process. The results provide a basis for understanding the unique features of molecular flux through connexins and other non-{beta}-barrel wide-pore channels.

biophysics↗

bioRxiv2019

A Membrane-Targeted Photoswitch Potently Modulates Neuronal Firing

Optical technologies allowing modulation of neuronal activity at high spatio-temporal resolution are becoming paramount in neuroscience. We engineered novel light-sensitive molecules by adding polar groups to a hydrophobic backbone containing azobenzene and azepane moieties. We demonstrate that the probes stably partition into the plasma membrane, with affinity for lipid rafts, and cause thinning of the bilayer through their trans-dimerization in the dark. In neurons pulse-labeled with the compound, light induces a transient hyperpolarization followed by a delayed depolarization that triggers action potential firing. The fast hyperpolarization is attributable to a light-dependent decrease in capacitance due to membrane relaxation that follows disruption of the azobenzene dimers. The physiological effects are persistent and can be evoked in vivo after labeling the mouse somatosensory cortex. These data demonstrate the possibility to trigger neural activity in vitro and in vivo by modulating membrane capacitance, without directly affecting ion channels or local temperature.

neuroscience↗