Helical Domain Research Articles

The proline-rich antimicrobial peptide Api137 disrupts large ribosomal subunit assembly and induces misfolding

The proline-rich antimicrobial designer peptide Api137 inhibits protein expression in bacteria by binding simultaneously to the ribosomal polypeptide exit tunnel and the release factor (RF), depleting the cellular RF pool and leading to ribosomal arrest at stop codons. This study investigates the additional effect of Api137 on the assembly of ribosomes using an Escherichia coli reporter strain expressing one ribosomal protein per 30S and 50S subunit tagged with mCherry and EGFP, respectively. Separation of cellular extracts derived from cells exposed to Api137 in a sucrose gradient reveals elevated levels of partially assembled and not fully matured precursors of the 50S subunit (pre-50S). High-resolution structures obtained by cryogenic electron microscopy demonstrate that a large proportion of pre-50S states are missing up to five proteins (uL22, bL32, uL29, bL23, and uL16) and have misfolded helices in 23S rRNA domain IV. These data suggest a second mechanism for Api137, wherein it disrupts 50S subunit assembly by inducing the formation of misfolded precursor particles potentially incapable of evolving into active ribosomes, suggesting a bactericidal mechanism.

MTOR Variants Activation Discovers PI3K-like Cryptic Pocket, Expanding Allosteric, Mutant-Selective Inhibitor Designs.

mTOR plays a crucial role in PI3K/AKT/mTOR signaling. We hypothesized that mTOR activation mechanisms driving oncogenesis can advise effective therapeutic designs. To test this, we combined cancer genomic analysis with extensive molecular dynamics simulations of mTOR oncogenic variants. We observed that conformational changes within mTOR kinase domain are associated with multiple mutational activation events. The mutations disturb the α-packing formed by the kαAL, kα3, kα9, kα9b, and kα10 helices in the kinase domain, creating cryptic pocket. Its opening correlates with opening of the catalytic cleft, including active site residues realignment, favoring catalysis. The cryptic pocket created by disrupted α-packing coincides with the allosteric pocket in PI3Kα can be harmoniously fitted by the PI3Kα allosteric inhibitor RLY-2608, suggesting that analogous drugs designed based on RLY-2608 can restore the packed α-structure, resulting in mTOR inactive conformation. Our results exemplify that knowledge of detailed kinase activation mechanisms can inform innovative allosteric inhibitor development.

Split Membrane: A New Model to Accelerate All-Atom MD Simulation of Phospholipid Bilayers.

All-atom molecular dynamics simulations are powerful tools for studying cell membranes and their interactions with proteins and other molecules. However, these processes occur on time scales determined by the diffusion rate of phospholipids, which are challenging to achieve in all-atom models. Here, we present a new all-atom model that accelerates lipid diffusion by splitting phospholipid molecules into head and tail groups. The bilayer structure is maintained by using external lateral potentials, which compensate for the lipid split. This split model enhances lateral lipid diffusion more than ten times, allowing faster and cheaper equilibration of large systems with different phospholipid types. The current model has been tested on membranes containing PSM, POPC, POPS, POPE, POPA, and cholesterol. We have also evaluated the interaction of the split model membranes with the Disheveled DEP domain and amphiphilic helix motif of the transcriptional repressor Opi1 as representative of peripheral proteins as well as the dimeric fragment of the epidermal growth factor receptor transmembrane domain and the Human A2A Adenosine of G protein-coupled receptors as representative of transmembrane proteins. The split model can predict the interaction sites of proteins and their preferred phospholipid type. Thus, the model could be used to identify lipid binding sites and equilibrate large membranes at an affordable computational cost.

Neurofilament Light Chain under the Lens of Structural Mass Spectrometry.

Neurofilament light chain (NfL) is an early nonspecific biomarker in neurodegenerative diseases and traumatic brain injury, indicating axonal damage. This work describes the detailed structural characterization of a selected primary calibrator with the potential to be used in future reference measurement procedure (RMP) development for the accurate quantification of NfL. As a part of the described workflow, the sequence, higher-order structure as well as solvent accessibility, and hydrogen-bonding profile were assessed under three different conditions in KPBS, artificial cerebrospinal fluid, and artificial cerebrospinal fluid in the presence of human serum albumin. The results revealed that NfL is a structurally heterogeneous protein, eliciting a large conformational flexibility. Its structural ensemble changed when it was diluted with an aqueous buffer versus a surrogate matrix, artificial cerebrospinal fluid (aCSF), and/or aCSF with human serum albumin. Various regions of protection and deprotection in the protein head, central helical, and tail domains that experienced altered solvent accessibility and conformational changes caused by different solvent conditions were identified. Moreover, interfacial residues, which may play a role in a potential direct interaction between NfL and human serum albumin, emerged from hydrogen-deuterium exchange mass spectrometry (HDX-MS). These data pinpointed distinct regions of the protein that may participate in such an interaction. Overall, critical quality attributes of a potential primary calibrator for NfL measurements are provided. These findings will ultimately inform ongoing biochemical and clinical assay development procedures and manufacturing practices, giving careful consideration during sample handling and method development.

Structural insight into the poly(3-hydroxybutyrate) hydrolysis by intracellular PHB depolymerase from Bacillus thuringiensis.

Poly((R)-3-hydroxybutyrate) (PHB) is a microbial biopolymer widely used in commercial biodegradable plastics. PHB degradation in cell is catalyzed by PHB depolymerase (PhaZ), which hydrolyzes the polyester into mono- and/or oligomeric (R)-3-hydroxylbutyrates (3HB). A novel intracellular PhaZ from Bacillus thuringiensis (BtPhaZ) was identified for potential applications in polymer biodegradation and 3HB production. Herein, we present the crystal structure of BtPhaZ at 1.42-Å resolution, making the first crystal structure for an intracellular PhaZ. BtPhaZ comprises a canonical α/β hydrolase catalytic domain and a unique α-helical cap domain. Despite lacking sequence similarity, BtPhaZ shares high structural homology with many α/β hydrolase members, exhibiting a similar active-site architecture. Alongside the most conserved superfamily signature, several new conserved signatures have been identified, contributing not only to the formations of the Ser-His-Asp catalytic triad and the oxyanion hole but also to the active-site conformation. The putative P-1 subsite appears to have limited space for accommodating only one 3HB-monomer, which may provide an explanation why the major hydrolytic product for BtPhaZ is monomeric form. Furthermore, a cluster of solvent-exposed hydrophobic residues in the helical cap domain forms an adsorption site for polymer-binding. Detailed structural comparisons reveal that various PhaZs employ distinct residues for the biopolymer-binding and hydrolysis.

Plasmodium falciparum surf4.1 in clinical isolates: From genetic variation and variant diversity to in silico design immunopeptides for vaccine development.

SURFINs protein family expressed on surface of both infected red blood cell and merozoite surface making them as interesting vaccine candidate for erythrocytic stage of malaria infection. In this study, we analyze genetic variation of Pfsurf4.1 gene, copy number variation, and frequency of SURFIN4.1 variants of P. falciparum in clinical isolates. In addition, secondary structure prediction and immunoinformatic were employed to identify immunogenic epitopes in humoral response as proposed vaccine candidates. Overall, our data demonstrate extensive polymorphism of SURFIN4.1 in both genetic and protein level. The surf4.1 gene showed extensive genetic variation with total of 447 polymorphic sites with maximum of three variants as well as singlet/triplet bases indels and mini/microsatellites in the coding sequence. The exon1 encoding extracellular region exhibited higher variation compared to exon2 which coding for intracellular domain. Interestingly, selective pressure was detected on both extracellular region (Var1 and Var2) as well as intracellular region (WRD2 and WRD3). Importantly, extensive full gene analysis suggests adenosine insertion at three key points nucleotide bases (nt 2409/2410, 3809/3810, and 4439/4440) of exon2 could lead to frameshift mutation resulted in four different SURFIN4.1 variants (TMs, WD1, WD2 and WD3). The SURFIN4.1 variant TMs was the most observed type with 67% frequency (51/76). Along with more than one copy number of surf4.1 gene was observed with frequency of 13% (9/70). Despite substantial polymorphism, analysis of relatedness within P. falciparum population using full coding sequence was able to group SURFIN4.1 protein into five distinct clades and reduced into four clades when using only exon1 coding sequence. Also, predicted secondary structure revealed conserved structure of five helix domains of extracellular region which similar among four SURFIN4.1 variant types. In addition, in silico design eight immunopeptides derived from SURFIN4.1, four of which are highly conserved and four of dimorphic epitopes, as potential vaccine candidates.

Cellular Function of a Biomolecular Condensate Is Determined by Its Ultrastructure.

Biomolecular condensates play key roles in the spatiotemporal regulation of cellular processes. Yet, the relationship between atomic features and condensate function remains poorly understood. We studied this relationship using the polar organizing protein Z (PopZ) as a model system, revealing how its material properties and cellular function depend on its ultrastructure. We revealed PopZ's hierarchical assembly into a filamentous condensate by integrating cryo-electron tomography, biochemistry, single-molecule techniques, and molecular dynamics simulations. The helical domain drives filamentation and condensation, while the disordered domain inhibits them. Phase-dependent conformational changes prevent interfilament contacts in the dilute phase and expose client binding sites in the dense phase. These findings establish a multiscale framework that links molecular interactions and condensate ultrastructure to macroscopic material properties that drive cellular function.

Functionally important components of the transcription elongation complex involved in Rho-dependent termination.

Bacterial transcription terminator, Rho is an RNA-dependent ATPase that terminates transcription. Several structures of pre-termination complexes of the Rho-transcription elongation complex (EC) revealed a static picture of components of the EC that come close to the nascent RNA-bound Rho, where many of the residues of EC reside≤10Å from the Rho residues. However, the in vitro-formed Rho-EC complexes do not reveal the in vivo Rho-EC dynamic interaction patterns during the termination process. Here we report synthetic defect analyses of various combinations of the mutations in RNAP β, β' and ω-subunits, NusA, NusG, and Rho proteins to delineate the functional network of this process. Several mutations in the β-flap and β'-Zn-finger and -Clamp helices domains of RNAP are synthetically defective in the presence of Rho mutants indicating functional involvement of these domains. Mutations in the NusA RNA-binding domains were synthetically defective with the Rho mutants suggesting its involvement. Our genetic analyses also revealed functional antagonisms between the ω-subunit of RNAP and the NusG-CTD during termination. We concluded that the regions surrounding the RNA exit channel, the RNA-binding domains of NusA, the RNAP ω-subunit, and NusG-CTD constitute a functional network with Rho just before the onset of in vivo Rho-dependent termination.

Alpha-helices as alignment reporters in residual dipolar coupling analysis of proteins.

Inclusion of residual dipolar couplings (RDCs) during the early rounds of protein structure determination requires use of a floating alignment tensor or knowledge of the alignment tensor strength and rhombicity. For proteins with interdomain motion, such analysis can falsely hide the presence of domain dynamics. We demonstrate for three proteins, maltotriose-ligated maltose binding protein (MBP), Ca2+-ligated calmodulin, and a monomeric N-terminal deletion mutant of the SARS-CoV-2 Main Protease, MPro, that good alignment tensor estimates of their domains can be obtained from RDCs measured for residues that are identified as α-helical based on their chemical shifts. The program, Helix-Fit, fits the RDCs to idealized α-helical coordinates, often yielding a comparable or better alignment tensor estimate than fitting to the actual high-resolution X-ray helix coordinates. The 13 helices of ligated MBP all show very similar alignment tensors, indicative of a high degree of order relative to one another. By contrast, while for monomeric MPro the alignment strengths of the five helices in the C-terminal helical domain (residues 200-306) are very similar, pointing to a well-ordered domain, the single α-helix Y54-I59 in the N-terminal catalytic domain (residues 10-185) aligns considerably weaker. This result indicates the presence of large amplitude motions of either Y54-I59 or of the entire N-terminal domain relative to the C-terminal domain, contrasting with the high degree of order seen in the native homodimeric structure.

Structural basis for Rab6 activation by the Ric1-Rgp1 complex

Rab GTPases act as molecular switches to regulate organelle homeostasis and membrane trafficking. Rab6 plays a central role in regulating cargo flux through the Golgi and is activated via nucleotide exchange by the Ric1-Rgp1 protein complex. Ric1-Rgp1 is conserved throughout eukaryotes but the structural and mechanistic basis for its function has not been established. Here we report the cryoEM structure of a Ric1-Rgp1‐Rab6 complex representing a key intermediate of the nucleotide exchange reaction. Ric1-Rgp1 interacts with the nucleotide-binding domain of Rab6 using an uncharacterized helical domain, which we establish as a RabGEF domain by identifying residues required for Rab6 activation. Unexpectedly, the complex uses an arrestin fold to interact with the Rab6 hypervariable domain, indicating that interactions with the unstructured C-terminal regions of Rab GTPases may be a common binding mechanism used by their activators. Collectively, our findings provide a detailed mechanistic understanding of regulated Rab6 activation at the Golgi.

The crystal and cryo-EM structures of PLCγ2 reveal dynamic interdomain recognitions in autoinhibition.

Phospholipase C gamma 2 (PLCγ2) plays important roles in cell signaling downstream of various membrane receptors. PLCγ2 contains a multidomain inhibitory region critical for its regulation, while it has remained unclear how these domains contribute to PLCγ2 activity modulation. Here we determined three structures of human PLCγ2 in autoinhibited states, which reveal dynamic interactions at the autoinhibition interface, involving the conformational flexibility of the Src homology 3 (SH3) domain in the inhibitory region, and its previously unknown interaction with a carboxyl-terminal helical domain in the core region. We also determined a structure of PLCγ2 bound to the kinase domain of fibroblast growth factor receptor 1 (FGFR1), which demonstrates the recognition of FGFR1 by the nSH2 domain in the inhibitory region of PLCγ2. Our results provide structural insights into PLCγ2 regulation that will facilitate future mechanistic studies to understand the entire activation process.

Molecular genetic and morphological characteristics of Micractinium thermotolerans and M. inermum (Trebouxiophyceae, Chlorophyta) from pyroclastic deposits of the Kamchatka Peninsula (Russia).

During the study of algal diversity in pyroclastic deposits of the Kamchatka Peninsula, Chlorella-like green algae strains VCA-72 and VCA-93 were isolated from samples collected from along the Baydarnaya river bed on the Shiveluch volcano in 2018 and at the outlet of thermal vapors along the edge of the caldera on the southern slope of the Gorely volcano in 2020. Identification of the strains was carried out within the framework of an integrative approach using microscopic and molecular genetic methods, including preliminary taxon identification, obtaining nucleotide sequences of the small subunit and the internal transcribed spacer rRNA, reconstruction of phylogenetic trees and secondary structures of the ITS1 and ITS2 rRNA regions. On the phylogenetic tree, strain VCA-93 was clustered in the Micractinium thermotolerans species clade. No differences were found when comparing the helical domain models of ITS1 and ITS2 in M. thermotolerans. Strain VCA-72 occupied a basal position in the M. inermum clade. The secondary structure patterns of the helices of strain VCA-72 were generally similar to those of M. inermum, but intraspecific variability was noted, mainly due to substitutions in the apical and lateral loops. Five hCBC substitutions were found in the helical regions of the studied M. inermum strains, while no CBC substitutions were found. A detailed analysis of morphology and life cycle allowed us to identify the characteristics of the cells in aging cultures: their size was significantly higher than in vegetative ones and they were pear-shaped, oval, and ellipsoidal with a shallow, wide constriction in the center. In addition, cells with colorless lipid droplets were detected in aging cultures of both species. The ability to synthesize and accumulate lipids indicates the great potential of the strains for the production of biodiesel fuel. A review of the habitats of previous and new findings allowed us to note the ecological plasticity of the studied species. The results obtained complement the information on the biogeography of the species: this is the first record of M. inermum for the territory of Russia, and that of M. thermotolerans, for the Kamchatka Peninsula.

Strain mediated transition between skyrmion and antiskyrmion in ferromagnetic thin films

Magnetic topological structures have attracted great attention due to their potential applications in memory and logic devices. Achieving the controllable transition between different magnetic topological structures is crucial for their application. Here, we develop a phase field model with strain-modulated Dzyaloshinskii-Moriya interaction (DMI) and predict the controllable transitions between skyrmion and antiskyrmion states in a ferromagnetic thin film through the application of different strains. It is found that the anisotropic DMI induced by anisotropic strains in the thin film plays an important role in the transitions between various magnetic structures, including skyrmion, antiskyrmion, single domain, and helical domain. Anisotropic DMI also has a significant impact on the chirality and deformation of magnetic topological structures, among which anisotropic DMI can cause anisotropic deformation of skyrmions and antiskyrmions. Furthermore, the formation mechanism of antiskyrmions is elucidated by decomposing the magnetization vectors into Bloch and Néel-type components based on the Lifshitz invariant. This work not only provides an insight into the dynamic behaviors of topological structures but also suggests a new method for controlling magnetic configurations through strain engineering.

Covalent adducts formed by the androgen receptor transactivation domain and small molecule drugs remain disordered.

Intrinsically disordered proteins are implicated in many human diseases. Small molecules that target the disordered androgen receptor transactivation domain have entered human trials for the treatment of castration-resistant prostate cancer. These molecules have been shown to react with cysteine residues of the androgen receptor transactivation domain and form covalent adducts under physiological conditions. It is currently unclear how covalent attachment of these molecules alters the conformational ensemble of the androgen receptor. Here, we utilize all-atom molecular dynamics computer simulations to simulate covalent adducts of the small molecule ligands EPI-002 and EPI-7170 bound to the disordered androgen receptor transactivation domain. Our simulations reveal that the conformational ensembles of androgen receptor transactivation domain covalent adducts are heterogeneous and disordered. We find that covalent attachment of EPI-002 and EPI-7170 increases the population of collapsed helical transactivation domain conformations relative to the populations observed in non-covalent binding simulations and we identify networks of protein-ligand interactions that stabilize collapsed conformations in covalent adduct ensembles. We compare the populations of protein-ligand interactions observed in covalent adduct ensembles to those observed in non-covalent ligand-bound ensembles and find substantial differences. Our results provide atomically detailed descriptions of covalent adducts formed by small molecules and an intrinsically disordered protein and suggest strategies for developing more potent covalent inhibitors of intrinsically disordered proteins.

Heterologous protein exposure and secretion optimization in Mycoplasma pneumoniae

The non-pathogenic Mycoplasma pneumoniae engineered chassis (Mycochassis) has demonstrated the ability to express therapeutic molecules in vitro and to be effective for treatment of lung infectious diseases in in vivo mouse models. However, the expression of heterologous molecules, whether secreted or exposed on the bacterial membrane has not been optimized to ensure sufficient secretion and/or exposure levels to exert a maximum in vivo biological effect. Here, we have improved the currently used secretion signal from MPN142 protein. We found that mutations at P1’ position of the signal peptide cleavage site do not abrogate secretion but affect it. Increasing hydrophobicity and mutations at the C-terminal of the signal peptide increases secretion. We tested different lipoprotein signal peptides as possible N-terminal protein anchoring motifs on the Mpn cell surface. Unexpectedly we found that these peptides exhibit variable retention and secretion rates of the protein, with some sequences behaving as full secretion motifs. This raises the question of the biological role of the lipobox motif traditionally thought to anchor membrane proteins without a helical transmembrane domain. These results altogether represent a step forward in chassis optimization, offering different sequences for secretion or membrane retention, which could be used to improve Mycochassis as a delivery vector, and broadening its therapeutic possibilities.

Computational Insights into Acrylamide Fragment Inhibition of SARS-CoV-2 Main Protease.

The pathogen of COVID-19, SARS-CoV-2, has caused a severe global health crisis. So far, while COVID-19 has been suppressed, the continuous evolution of SARS-CoV-2 variants has reduced the effectiveness of vaccines such as mRNA-1273 and drugs such as Remdesivir. To uphold the effectiveness of vaccines and drugs prior to potential coronavirus outbreaks, it is necessary to explore the underlying mechanisms between biomolecules and nanodrugs. The experimental study reported that acrylamide fragments covalently attached to Cys145, the main protease enzyme (Mpro) of SARS-CoV-2, and occupied the substrate binding pocket, thereby disrupting protease dimerization. However, the potential mechanism linking them is unclear. The purpose of this work is to complement and validate experimental results, as well as to facilitate the study of novel antiviral drugs. Based on our experimental studies, we identified two acrylamide fragments and constructed corresponding protein-ligand complex models. Subsequently, we performed molecular dynamics (MD) simulations to unveil the crucial interaction mechanisms between these nanodrugs and SARS-CoV-2 Mpro. This approach allowed the capture of various binding conformations of the fragments on both monomeric and dimeric Mpro, revealing significant conformational dissociation between the catalytic and helix domains, which indicates the presence of allosteric targets. Notably, Compound 5 destabilizes Mpro dimerization and acts as an effective inhibitor by specifically targeting the active site, resulting in enhanced inhibitory effects. Consequently, these fragments can modulate Mpro's conformational equilibrium among extended monomeric, compact, and dimeric forms, shedding light on the potential of these small molecules as novel inhibitors against coronaviruses. Overall, this research contributes to a broader understanding of drug development and fragment-based approaches in antiviral covalent therapeutics.

Fungus-derived opine enhances plant photosynthesis

IntroductionPlant-fungal interactions stimulate endophytic fungi to produce a plethora of metabolites that enhance plant growth and improve stress resistance. Opines, naturally occurring compounds formed through the condensation of amino acids with α-keto acids or sugars, have diverse biological functions and are mainly present in bacteria. Interestingly, investigations have revealed the presence of opine synthases (OSases) in fungal species as well, and their functions are yet to be studied. ObjectivesThe objective of this study is to investigate the occurrence of OSases in fungal species, identify their products, and characterize the potential biological activity of the metabolites. MethodsWe identified a putative class of OSases in fungi through sequence similarity network (SSN) analysis. The function of these enzymes was elucidated using methods including protein heterologous expression, in vitro biochemical characterization, in vivo gene knock-out, as well as product isolation and identification. Additionally, we conducted plant activity testing on the secondary metabolites through foliar spraying and performed transcriptomic analysis to uncover their functions. ResultsA quarter of the PF18631 family members, which contain the C-terminal helical bundle domain of cucumopine synthase, are derived from endophytic fungi. Some of these enzymes catalyze the synthesis of tryptopine A (1-acetyl-3-carboxy-β-carboline) by condensing L-tryptophan and methylglyoxal. The tryptopine A can act as a growth regulator, promoting plant growth and transcriptionally reprogramming photosynthesis-related pathways, while enhancing the rate of plant photosynthesis by 25%. ConclusionThe findings of this study suggest that tryptopine A plays a crucial role as a signaling molecule in the establishment and maintenance of mutualistic associations between endophytic fungi and host plants, thereby broaden our comprehension of fungal-plant symbiosis.

Host species-specific activity of the poxvirus PKR inhibitors E3 and K3 mediate host range function.

The antiviral protein kinase R (PKR) is activated by viral double-stranded RNA and phosphorylates translation initiation factor eIF2α, thereby inhibiting translation and virus replication. Most poxviruses contain two PKR inhibitors, called E3 and K3 in vaccinia virus (VACV), which are determinants of viral host range. The prevailing model for E3 function is that it inhibits PKR through the non-specific sequestration of double-stranded (ds) RNA. Our data revealed that Syrian hamster PKR was resistant to E3, which is at odds with the sequestration model. However, Syrian hamster PKR was still sensitive to K3 inhibition. In contrast, Armenian hamster PKR showed opposite sensitivities, being sensitive to E3 and resistant to K3 inhibition. Mutational analyses of hamster PKRs showed that sensitivity to E3 inhibition was largely determined by the region linking the dsRNA-binding domains and the kinase domain of PKR, whereas two amino acid residues in the kinase domain (helix αG) determined sensitivity to K3. The expression of PKRs in congenic cells showed that Syrian hamster PKR containing the two Armenian hamster PKR residues in helix αG was resistant to wild-type VACV infection and that cells expressing either hamster PKR recapitulated the phenotypes observed in species-derived cell lines. The observed resistance of Syrian hamster PKR to E3 explains its host range function and challenges the paradigm that dsRNA-binding PKR inhibitors mainly act by the sequestration of dsRNA.IMPORTANCEThe molecular mechanisms that govern the host range of viruses are incompletely understood. We show that the host range functions of E3 and K3, two host range factors from vaccinia virus, are a result of species-specific interactions with the antiviral protein kinase R (PKR) and that PKR from closely related species displayed dramatic differences in their sensitivities to these viral inhibitors. The current model for E3-mediated PKR inhibition is that E3 non-specifically sequesters double-stranded (ds) RNA to prevent PKR activation. This model does not predict species-specific sensitivity to E3; therefore, our data suggest that the current model is incomplete and that dsRNA sequestration is not the primary mechanism for E3 activity.

Structural Basis for Voltage Gating and Dalfampridine Binding in the Shaker Potassium Channel.

The generation and propagation of action potentials in neurons depend on the coordinated activation of voltage-dependent sodium and potassium channels. Potassium channels of the Shaker family regulate neuronal excitability through voltage-dependent opening and closing of their ion conduction pore. This family of channels is an important therapeutic target, particularly in multiple sclerosis where the inhibitor dalfampridine (4-aminopyridine) is used to improve nerve conduction. The molecular details of how the voltage sensor domain drives opening of the pore domain has been limited by the lack of closed-state structures, also impairing the search for novel drugs. Using AlphaFold2-based conformational sampling methods we identify a structural model for the closed Shaker potassium channel where movement of the voltage sensor drives the opening trough interactions between the S4-S5 linker and S6 helix. We show experimentally that breakage of a backbone hydrogen bond is a critical part of the activation pathway. Through docking we identify a hydrophobic cavity formed by the pore domain helices that binds dalfampridine in the closed state. Our results demonstrate how voltage sensor movement drives pore opening and provide a structural framework for developing new therapeutic agents targeting the closed state. We anticipate this work will enable structure-based drug design efforts focused on state-dependent modulation of voltage-gated ion channels for the treatment of neurological disorders.

A novel CLRN2 variant: expanding the mutation spectrum and its critical role in isolated hearing impairment.

Biallelic variants in the CLRN2 gene have been reported to cause autosomal recessive profound hearing impairment in humans. CLRN2 belongs to the clarin gene family that encodes a tetraspan protein that contains a cytosolic N-terminus, multiple helical transmembrane domains, and an endoplasmic reticulum membrane retention signal, TKGH, in the C-terminus. The encoded protein may be important in development and homeostasis of the inner ear and retina. Here, we present a consanguineous family suffering from autosomal recessive non-syndromic profound hearing impairment (HI). We employed state of the art Whole exome sequencing (WES), Sanger sequencing followed by routine bioinformatics filtration steps and homology modeling to elucidate the effect of mutation at the protein level. ES followed by Sanger sequencing revealed a novel homozygous nonsense variant in the CLRN2 gene [c.414C > A; p.Cys138*]. Furthermore, insilico protein modeling of the wildtype and mutated version of the CLRN2 protein revealed large-scale changes that predict to compromise the routine normal function of the protein. Our finding further extends the mutations spectrum of CLRN2 gene and confirms its important role in hearing homeostasis and with developmental disorder in humans.