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The voltage-dependent anion channel (VDAC) is a crucial mitochondrial protein that facilitates ion and metabolite exchange between mitochondria and the cytosol. Initially characterized over three decades ago, the structure of VDAC-1 was resolved in 2008, revealing a novel β-barrel protein architecture. This study presents the first room-temperature crystal structure of mouse VDAC-1 (mVDAC-1), which is a significant step toward understanding the channel's gating mechanism. The new structure, obtained at a 3.3 Å resolution, demonstrates notable differences from the previously determined cryogenic structure, particularly in the loop regions, which may be critical for the transition between the 'open' and 'closed' states of VDAC-1. Comparative analysis of the root-mean-square deviation (R.M.S.D.) and B-factors between the cryogenic and room-temperature structures suggests that these conformational differences, although subtle, are important for VDAC's functional transitions. The application of electric field-stimulated X-ray crystallography (EF-X) is proposed as a future direction to resolve the 'closed' state of VDAC-1 by inducing voltage-driven conformational changes in order to elucidate the dynamic gating mechanism of VDAC-1. Our findings have profound implications for understanding the molecular basis of VDAC's role in mitochondrial function and its regulation under physiological conditions.

Sodium-dependent glucose transporters (SGLTs) couple a downhill Na+ ion gradient to actively transport sugars. Here, we investigate the impact of the membrane potential on vSGLT structure and function using sugar uptake assays, double electron-electron resonance (DEER), electrostatic calculations, and kinetic modeling. Negative membrane potentials, as present in all cell types, shift the conformational equilibrium of vSGLT towards an outward-facing conformation, leading to increased sugar transport rates. Electrostatic calculations identify gating charge residues responsible for this conformational shift that when mutated reduce galactose transport and eliminate the response of vSGLT to potential. Based on these findings, we propose a comprehensive framework for sugar transport via vSGLT, where the cellular membrane potential facilitates resetting of the transporter after cargo release. This framework holds significance not only for SGLTs but also for other transporters and channels.

BackgroundWe aimed to define the clinical and variant spectrum and to provide novel molecular insights into the DHX30-associated neurodevelopmental disorder.MethodsClinical and genetic data from affected individuals were collected through Facebook-based family support group, GeneMatcher, and our network of collaborators. We investigated the impact of novel missense variants with respect to ATPase and helicase activity, stress granule (SG) formation, global translation, and their effect on embryonic development in zebrafish. SG formation was additionally analyzed in CRISPR/Cas9-mediated DHX30-deficient HEK293T and zebrafish models, along with in vivo behavioral assays.ResultsWe identified 25 previously unreported individuals, ten of whom carry novel variants, two of which are recurrent, and provide evidence of gonadal mosaicism in one family. All 19 individuals harboring heterozygous missense variants within helicase core motifs (HCMs) have global developmental delay, intellectual disability, severe speech impairment, and gait abnormalities. These variants impair the ATPase and helicase activity of DHX30, trigger SG formation, interfere with global translation, and cause developmental defects in a zebrafish model. Notably, 4 individuals harboring heterozygous variants resulting either in haploinsufficiency or truncated proteins presented with a milder clinical course, similar to an individual harboring a de novo mosaic HCM missense variant. Functionally, we established DHX30 as an ATP-dependent RNA helicase and as an evolutionary conserved factor in SG assembly. Based on the clinical course, the variant location, and type we establish two distinct clinical subtypes. DHX30 loss-of-function variants cause a milder phenotype whereas a severe phenotype is caused by HCM missense variants that, in addition to the loss of ATPase and helicase activity, lead to a detrimental gain-of-function with respect to SG formation. Behavioral characterization of dhx30-deficient zebrafish revealed altered sleep-wake activity and social interaction, partially resembling the human phenotype.ConclusionsOur study highlights the usefulness of social media to define novel Mendelian disorders and exemplifies how functional analyses accompanied by clinical and genetic findings can define clinically distinct subtypes for ultra-rare disorders. Such approaches require close interdisciplinary collaboration between families/legal representatives of the affected individuals, clinicians, molecular genetics diagnostic laboratories, and research laboratories.

Although the role of hydrophilic antioxidants in the development of hepatic insulin resistance and nonalcoholic fatty liver disease has been well studied, the role of lipophilic antioxidants remains poorly characterized. A known lipophilic hydrogen peroxide scavenger is bilirubin, which can be oxidized to biliverdin and then reduced back to bilirubin by cytosolic biliverdin reductase. Oxidation of bilirubin to biliverdin inside mitochondria must be followed by the export of biliverdin to the cytosol, where biliverdin is reduced back to bilirubin. Thus, the putative mitochondrial exporter of biliverdin is expected to be a major determinant of bilirubin regeneration and intracellular hydrogen peroxide scavenging. Here, we identified ABCB10 as a mitochondrial biliverdin exporter. ABCB10 reconstituted into liposomes transported biliverdin, and ABCB10 deletion caused accumulation of biliverdin inside mitochondria. Obesity with insulin resistance up-regulated hepatic ABCB10 expression in mice and elevated cytosolic and mitochondrial bilirubin content in an ABCB10-dependent manner. Revealing a maladaptive role of ABCB10-driven bilirubin synthesis, hepatic ABCB10 deletion protected diet-induced obese mice from steatosis and hyperglycemia, improving insulin-mediated suppression of glucose production and decreasing lipogenic SREBP-1c expression. Protection was concurrent with enhanced mitochondrial function and increased inactivation of PTP1B, a phosphatase disrupting insulin signaling and elevating SREBP-1c expression. Restoration of cellular bilirubin content in ABCB10 KO hepatocytes reversed the improvements in mitochondrial function and PTP1B inactivation, demonstrating that bilirubin was the maladaptive effector linked to ABCB10 function. Thus, we identified a fundamental transport process that amplifies intracellular bilirubin redox actions, which can exacerbate insulin resistance and steatosis in obesity.

Smith-Kingsmore syndrome (SKS) is a rare neurodevelopmental disorder characterized by macrocephaly/megalencephaly, developmental delay, intellectual disability, hypotonia, and seizures. It is caused by dominant missense mutations in MTOR. The pathogenicity of novel variants in MTOR in patients with neurodevelopmental disorders can be difficult to determine and the mechanism by which variants cause disease remains poorly understood. We report 7 patients with SKS with 4 novel MTOR variants and describe their phenotypes. We perform in vitro functional analyses to confirm MTOR activation and interrogate disease mechanisms. We complete structural analyses to understand the 3D properties of pathogenic variants. We examine the accuracy of relative accessible surface area, a quantitative measure of amino acid side-chain accessibility, as a predictor of MTOR variant pathogenicity. We describe novel clinical features of patients with SKS. We confirm MTOR Complex 1 activation and identify MTOR Complex 2 activation as a new potential mechanism of disease in SKS. We find that pathogenic MTOR variants disproportionately cluster in hotspots in the core of the protein, where they disrupt alpha helix packing due to the insertion of bulky amino acid side chains. We find that relative accessible surface area is significantly lower for SKS-associated variants compared to benign variants. We expand the phenotype of SKS and demonstrate that additional pathways of activation may contribute to disease. Incorporating 3D properties of MTOR variants may help in pathogenicity classification. We hope these findings may contribute to improving the precision of care and therapeutic development for individuals with SKS.

Sodium-dependent glucose transporters (SGLTs) exploit sodium gradients to transport sugars across the plasma membrane. Due to their role in renal sugar reabsorption, SGLTs are targets for the treatment of type 2 diabetes. Current therapeutics are phlorizin derivatives that contain a sugar moiety bound to an aromatic aglycon tail. Here, we develop structural models of human SGLT1/2 in complex with inhibitors by combining computational and functional studies. Inhibitors bind with the sugar moiety in the sugar pocket and the aglycon tail in the extracellular vestibule. The binding poses corroborate mutagenesis studies and suggest a partial closure of the outer gate upon binding. The models also reveal a putative Na+ binding site in hSGLT1 whose disruption reduces the transport stoichiometry to the value observed in hSGLT2 and increases inhibition by aglycon tails. Our work demonstrates that subtype selectivity arises from Na+-regulated outer gate closure and a variable region in extracellular loop EL5.

Proteins possess a complex and dynamic structure, which is influenced by external signals and may change as they perform their biological functions. We present an optical approach, distance-encoding photoinduced electron transfer (DEPET), capable of the simultaneous study of protein structure and function. An alternative to FRET-based methods, DEPET is based on the quenching of small conjugated fluorophores by photoinduced electron transfer: a reaction that requires contact of the excited fluorophore with a suitable electron donor. This property allows DEPET to exhibit exceptional spatial and temporal resolution capabilities in the range pertinent to protein conformational change. We report the first implementation of DEPET on human large-conductance K+ (BK) channels under voltage clamp. We describe conformational rearrangements underpinning BK channel sensitivity to electrical excitation, in conducting channels expressed in living cells. Finally, we validate DEPET in synthetic peptide length standards, to evaluate its accuracy in measuring sub- and near-nanometer intramolecular distances.

This study solved structures of the glutamate-gated chloride channel (GluCl), a Cys-loop receptor from C. elegans, in an apo, closed state and in a lipid-bound state -- comparison of these structures with a previously published structure of GluCl in an ivermectin-bound state reveals what conformational changes probably occur as this membrane protein transitions from the closed/resting state towards an open/activated state. Resting-to-activated state of a Cys-loop receptor Depending on their ligand and ion selectivity, Cys-loop receptors are neurotransmitter-activated ion channels that mediate either excitatory or inhibitory neurotransmission. In this paper the authors solve the structures of the glutamate-gated chloride channel (GluCl), a Cys-loop receptor from Caenorhabditis elegans, in an apo or closed state and in a lipid-bound state. Comparison of these structures with the previously published structure of GluCl in an ivermectin-bound state reveals the conformational changes involved as this membrane protein transitions between the closed/resting and an open/activated state. Cys-loop receptors are neurotransmitter-gated ion channels that are essential mediators of fast chemical neurotransmission and are associated with a large number of neurological diseases and disorders, as well as parasitic infections.sup.1,2,3,4. Members of this ion channel superfamily mediate excitatory or inhibitory neurotransmission depending on their ligand and ion selectivity. Structural information for Cys-loop receptors comes from several sources including electron microscopic studies of the nicotinic acetylcholine receptor.sup.5, high-resolution X-ray structures of extracellular domains.sup.6 and X-ray structures of bacterial orthologues.sup.7,8,9,10. In 2011 our group published structures of the Caenorhabditis elegans glutamate-gated chloride channel (GluCl) in complex with the allosteric partial agonist ivermectin, which provided insights into the structure of a possibly open state of a eukaryotic Cys-loop receptor, the basis for anion selectivity and channel block, and the mechanism by which ivermectin and related molecules stabilize the open state and potentiate neurotransmitter binding.sup.11. However, there remain unanswered questions about the mechanism of channel opening and closing, the location and nature of the shut ion channel gate, the transitions between the closed/resting, open/activated and closed/desensitized states, and the mechanism by which conformational changes are coupled between the extracellular, orthosteric agonist binding domain and the transmembrane, ion channel domain. Here we present two conformationally distinct structures of C. elegans GluCl in the absence of ivermectin. Structural comparisons reveal a quaternary activation mechanism arising from rigid-body movements between the extracellular and transmembrane domains and a mechanism for modulation of the receptor by phospholipids., Author(s): Thorsten Althoff [sup.1] [sup.4] , Ryan E. Hibbs [sup.1] [sup.4] , Surajit Banerjee [sup.2] , Eric Gouaux [sup.1] [sup.3] Author Affiliations: (1) Vollum Institute, Oregon Health & Science University, [...]

Studies on membrane vesicles from the kidney of Leucoraja erinacea suggested the sole presence of a sodium-D-glucose cotransporter type 1 involved in renal D-glucose reabsorption. For molecular characterization of this transport system, an mRNA library was screened with primers directed against conserved regions of human sglt1. A cDNA was cloned whose nucleotide and derived amino acid sequence revealed high homology to sodium glucose cotransporter 1 (SGLT1). Xenopus laevis oocytes injected with the respective cRNA showed sodium-dependent high-affinity uptake of D-glucose. Many positions considered functionally essential for sodium glucose cotransporter 1 (SGLT1) are also found in the skate protein. High conservation preferentially in transmembrane helices and small linking loops suggests early appearance and continued preservation of these regions. Larger loops, especially loop 13, which is associated with phlorizin binding, were more variable, as is the interaction with the specific inhibitor in various species. To study the intrarenal distribution of the transporter, a skate SGLT1-specific antibody was generated. In cryosections of skate kidney, various nephron segments could be differentiated by lectin staining. Immunoreaction with the antibody was observed in the proximal tubule segments PIa and PIIa, the early distal tubule, and the collecting tubule. Thus Leucoraja, in contrast to the mammalian kidney, employs only SGLT1 to reabsorb D-glucose in the early, as well as in the late segments of the proximal tubule and probably also in the late distal tubule (LDT). Thereby, it differs also partly from the kidney of the close relative Squalus acanthias, which uses SGLT2 in more distal proximal tubule segments but shows also expression in the later nephron parts. sodium glucose cotransporter 1; intrarenal localization; cloning; fish; elasmobranch doi:10.1152/ajpregu.00454.2006

Using primers against conserved regions of mammalian [Na.sup.+]-D-glucose cotransporters (SGLT), a cDNA wits cloned from the kidney of spiny dogfish shark (Squalus acanthias). On the basis of comparison of amino acid sequence, membrane topology, and putative glycosylation and phosphorylation sites, the cDNA could be shown to belong to the family of sglt genes. Indeed, [Na.sup.+]-dependent D-glucose uptake could be demonstrated after expression of the gene in Xenopus laevis oocytes. In a dendrogram, the SGLT from shark kidney hits a high homology to the mammalian SGLT2. Computer analysis revealed that the elasmobranch protein is most similar to the mammalian proteins in the transmembrane regions and contains already all the amino acids identified to be functionally important, suggesting early conservation during evolution. Extramembraneous loops show larger variations. This holds especially for loop 13, which has been implied its a phlorizin-binding domain. Antibodies were generated and the intrarenal distribution of the SGLT was studied in cryosections. In parallel, the nephron segments were identified by lectins. Positive immunoreactions were found in the proximal tubule in the early parts PIa and PIb and the late segment PIIb. The large PIIa segment of the proximal tubule showed no reaction. In contrast to the mammalian kidney also the late distal tubule, the collecting tubule, and the collecting duct showed immunoreactivity. The molecular information confirms previous vesicle studies in which a low affinity SGLT with a low stoichiometry has been observed and supports the notion of a similarity of the shark kidney SGLT to the mammalian SGLT2. Despite its presence in the late parts of the nephron, the absence of SGLT in the major part of the proximal tubule, the relatively low affinity, and in particular the low stoichiometry might explain the lack of a [T.sub.m] for D-glucose in the shark kidney. sodium-glucose cotransporter; intrarenal localization; cloning; fish; shark; elasmobranch

The respiratory chain in the inner mitochondrial membrane contains three large multi-enzyme complexes that together establish the proton gradient for ATP synthesis, and assemble into a supercomplex. A 19-Å 3D map of the 1.7-MDa amphipol-solubilized supercomplex I1III2IV1 from bovine heart obtained by single-particle electron cryo-microscopy reveals an amphipol belt replacing the membrane lipid bilayer. A precise fit of the X-ray structures of complex I, the complex III dimer, and monomeric complex IV indicates distances of 13 nm between the ubiquinol-binding sites of complexes I and III, and of 10-11 nm between the cytochrome c binding sites of complexes III and IV. The arrangement of respiratory chain complexes suggests two possible pathways for efficient electron transfer through the supercomplex, of which the shorter branch through the complex III monomer proximal to complex I may be preferred. [ABSTRACT FROM AUTHOR]

Studies on membrane vesicles from the kidney of Leucoraja erinacea suggested the sole presence of a sodium-D-glucose cotransporter type 1 involved in renal D-glucose reabsorption. For molecular characterization of this transport system, an mRNA library was screened with primers directed against conserved regions of human sglt1. A cDNA was cloned whose nucleotide and derived amino acid sequence revealed high homology to sodium glucose cotransporter 1 (SGLT1). Xenopus Iaevis oocytes injected with the respective cRNA showed sodium-dependent high-affinity uptake of D-glucose. Many positions considered functionally essential for sodium glucose cotransporter 1 (SGLT1) are also found in the skate protein. High conservation preferentially in transmembrane helices and small linking loops suggests early appearance and continued preservation of these regions. Larger loops, especially loop 13, which is associated with phlorizin binding, were more variable, as is the interaction with the specific inhibitor in various species. To study the intrarenal distribution of the transporter, a skate SGLT1-specific antibody was generated. In cryo-sections of skate kidney, various nephron segments could be differentiated by lectin staining. Immunoreaction with the antibody was observed in the proximal tubule segments PIa and PIIa, the early distal tubule, and the collecting tubule. Thus Leucoraja, in contrast to the mammalian kidney, employs only SGLT1 to reabsorb D-glucose in the early, as well as in the late segments of the proximal tubule and probably also in the late distal tubule (LDT). Thereby, it differs also partly from the kidney of the close relative Squalus acanthias, which uses SGLT2 in more distal proximal tubule segments but shows also expression in the later nephron parts. [ABSTRACT FROM AUTHOR]

Using primers against con- served regions of mammalian Na+-n-glucose cotransporters (SGLT), a cDNA was cloned from the kidney of spiny dogfish shark (Squalus acanthias). On the basis of comparison of amino acid sequence, membrane topology, and putative glycosylation and phosphorylation sites, the cDNA could be shown to belong to the family of sglt genes. Indeed, Na+-dependent D-glucose uptake could be demonstrated after expression of the gene in Xenopus laevis oocytes. In a dendrogram, the SGLT from shark kidney has a high homology to the mammalian SGLT2. Computer analysis revealed that the elasmobranch protein is most similar to the mammalian proteins in the transmembrane regions and contains already all the amino acids identified to be functionally important, suggesting early conservation during evolution. Extramembraneous loops show larger variations. This holds especially for loop 13, which has been implied as a phlorizin-binding domain. Antibodies were generated and the intrarenal distribution of the SGLT was studied in cryosections. In parallel, the nephron segments were identified by lectins. Positive immunoreactions were found in the proximal tubule in the early parts PIa and PIb and the late segment PIIb. The large PIIa segment of the proximal tubule showed no reaction. In contrast to the mammalian kidney also the late distal tubule, the collecting tubule, and the collecting duct showed immunoreactivity. The molecular information confirms previous vesicle studies in which a low affinity SGLT with a low stoichiometry has been observed and supports the notion of a similarity of the shark kidney SGLT to the mammalian SGLT2. Despite its presence in the late parts of the nephron, the absence of SGLT in the major part of the proximal tubule, the relatively low affinity, and in particular the low stoichiometry might explain the lack of a Tm for D-glucose in the shark kidney. [ABSTRACT FROM AUTHOR]

Sodium-dependent glucose transporters (SGLTs) exploit sodium gradients to transport sugars across the plasma membrane. Due to their role in renal sugar reabsorption, SGLTs are targets for the treatment of type 2 diabetes. Current therapeutics are phlorizin derivatives that contain a sugar moiety bound to an aromatic aglycon tail. Here, we develop structural models of human SGLT1/2 in complex with inhibitors by combining computational and functional studies. Inhibitors bind with the sugar moiety in the sugar pocket and the aglycon tail in the extracellular vestibule. The binding poses corroborate mutagenesis studies and suggest a partial closure of the outer gate upon binding. The models also reveal a putative Na+ binding site in hSGLT1 whose disruption reduces the transport stoichiometry to the value observed in hSGLT2 and increases inhibition by aglycon tails. Our work demonstrates that subtype selectivity arises from Na+-regulated outer gate closure and a variable region in extracellular loop EL5. Sodium-dependent glucose transporters (SGLTs) transport sugars across the plasma membrane and play important roles in renal sugar reabsorption. Here authors develop structural models of human SGLT1/2 (hSGLT1/2) in complex with inhibitors which helps to understand inhibitor subtype selectivity. [ABSTRACT FROM AUTHOR]