Cryo-EM structure of the human glucose transporter GLUT7.

GLUT7 is a Class II glucose transporter predominantly expressed at the apical membrane of enterocytes in the small intestine. Here, we report the cryo-EM structure of nanodisc-reconstituted human GLUT7 in the apo state at 3.3 Å resolution.

Discovery of Triazolopyrimidine Derivatives as Selective P2X3 Receptor Antagonists Binding to an Unprecedented Allosteric Site as Evidenced by Cryo-Electron Microscopy.

The P2X3 receptor (P2X3R), an ATP-gated cation channel predominantly expressed in C- and Aδ-primary afferent neurons, has been proposed as a drug target for neurological inflammatory diseases, e.g., neuropathic pain, and chronic cough.

Activation mechanism and novel binding sites of the BK channel activator CTIBD.

The large-conductance calcium-activated potassium (BK) channel, which is crucial for urinary bladder smooth muscle relaxation, is a potential target for overactive bladder treatment. Our prior work unveiled CTIBD as a promising BK channel activator, altering and This study investigates CTIBD's activation mechanism, revealing its independence from the Ca and membrane voltage sensing of the BK channel. Cryo-electron microscopy disclosed that two CTIBD molecules bind to hydrophobic regions on the extracellular side of the lipid bilayer.

Cryo-EM structure of cadmium-bound human ABCB6.

ATP-binding cassette transporter B6 (ABCB6), a protein essential for heme biosynthesis in mitochondria, also functions as a heavy metal efflux pump. Here, we present cryo-electron microscopy structures of human ABCB6 bound to a cadmium Cd(II) ion in the presence of antioxidant thiol peptides glutathione (GSH) and phytochelatin 2 (PC2) at resolutions of 3.2 and 3.

Yeast lunapark regulates the formation of -Sey1p complexes for homotypic ER membrane fusion.

The endoplasmic reticulum (ER) consists of the nuclear envelope and a connected peripheral network of tubules and interspersed sheets. The structure of ER tubules is generated and maintained by various proteins, including reticulons, DP1/Yop1p, atlastins, and lunapark. Reticulons and DP1/Yop1p stabilize the high membrane curvature of ER tubules, and atlastins mediate homotypic membrane fusion between ER tubules; however, the exact role of lunapark remains poorly characterized.

W546 stacking disruption traps the human porphyrin transporter ABCB6 in an outward-facing transient state.

Human ATP-binding cassette transporter subfamily B6 (ABCB6) is a mitochondrial ATP-driven pump that translocates porphyrins from the cytoplasm into mitochondria for heme biosynthesis. Within the transport pathway, a conserved aromatic residue W546 located in each monomer plays a pivotal role in stabilizing the occluded conformation via π-stacking interactions. Herein, we employed cryo-electron microscopy to investigate the structural consequences of a single W546A mutation in ABCB6, both in detergent micelles and nanodiscs.

Discovery of antiviral SARS-CoV-2 main protease inhibitors by structure-guided hit-to-lead optimization of carmofur.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (M) has been targeted for the development of anti-SARS-CoV-2 agents against COVID-19 infection because M processes essential viral polyproteins and plays a key role in SARS-CoV-2 replication. In this study, we report the development of novel SARS-CoV-2 M inhibitors derived from carmofur, a previously identified compound that has shown moderate potency as a covalent inhibitor of SARS-CoV-2 M. To employ a structure-guided drug design strategy, a putative intact binding mode of carmofur at catalytic active site of M was initially predicted by docking simulation.

The lysosomal transporter TAPL has a dual role as peptide translocator and phosphatidylserine floppase.

TAPL is a lysosomal ATP-binding cassette transporter that translocates a broad spectrum of polypeptides from the cytoplasm into the lysosomal lumen. Here we report that, in addition to its well-known role as a peptide translocator, TAPL exhibits an ATP-dependent phosphatidylserine floppase activity that is the possible cause of its high basal ATPase activity and of the lack of coupling between ATP hydrolysis and peptide efflux. We also present the cryo-EM structures of mouse TAPL complexed with (i) phospholipid, (ii) cholesteryl hemisuccinate (CHS) and 9-mer peptide, and (iii) ADP·BeF.

Structural Insights into Porphyrin Recognition by the Human ATP-Binding Cassette Transporter ABCB6.

Human ABCB6 is an ATP-binding cassette transporter that regulates heme biosynthesis by translocating various porphyrins from the cytoplasm into the mitochondria. Here we report the cryo-electron microscopy (cryo-EM) structures of human ABCB6 with its substrates, coproporphyrin III (CPIII) and hemin, at 3.5 and 3.

Eltrombopag as an Allosteric Inhibitor of the METTL3-14 Complex Affecting the mA Methylation of RNA in Acute Myeloid Leukemia Cells.

NA-methyladenosine (mA) post-transcriptional modification, the most abundant internal RNA modification, is catalyzed by the METTL3-14 methyltransferase complex. Recently, attention has been drawn to the METTL3-14 complex regarding its significant roles in the pathogenesis of acute myeloid leukemia (AML), attracting the potential of novel therapeutic targets for the disease. Herein, we report the identification and characterization of eltrombopag as a selective allosteric inhibitor of the METTL3-14 complex.

A novel in-frame GFAP p.E138_L148del mutation in Type II Alexander disease with atypical phenotypes.

Alexander disease (AxD) is a neurodegenerative astrogliopathy caused by mutation in the glial fibrillary acidic protein (GFAP) gene. A 42-year-old Korean man presented with temporary gait disturbance and psychiatric regression after a minor head trauma in the absence of bulbar symptoms and signs. Magnetic resonance images of the brain and spinal cord showed significant atrophy of the medulla oblongata and the entire spinal cord as well as contrast-enhanced T2 hypointensity in the basal ganglia.

Structural insights into novel mechanisms of inhibition of the major β-carbonic anhydrase CafB from the pathogenic fungus Aspergillus fumigatus.

In fungi the β-class of carbonic anhydrases (β-CAs) are zinc metalloenzymes that are essential for growth, survival, differentiation, and virulence. Aspergillus fumigatus is the most important pathogen responsible for invasive aspergillosis and possesses two major β-CAs, CafA and CafB. Recently we reported the biochemical characterization and 1.

Crystal Structure of β-Carbonic Anhydrase CafA from the Fungal Pathogen .

The β-class of carbonic anhydrases (β-CAs) are zinc metalloenzymes widely distributed in the fungal kingdom that play essential roles in growth, survival, differentiation, and virulence by catalyzing the reversible interconversion of carbon dioxide (CO) and bicarbonate (HCO). Herein, we report the biochemical and crystallographic characterization of the β-CA CafA from the fungal pathogen , the main causative agent of invasive aspergillosis. CafA exhibited apparent CO hydration activity in neutral to weak alkaline conditions, but little activity at acidic pH.

The Minimal Effect of Linker Length for Fatty Acid Conjugation to a Small Protein on the Serum Half-Life Extension.

Conjugation of serum albumin or one of its ligands (such as fatty acid) has been an effective strategy to prolong the serum half-lives of drugs via neonatal Fc receptor (FcRn)-mediated recycling of albumin. So far, fatty acid (FA) has been effective in prolonging the serum half-lives for therapeutic peptides and small proteins, but not for large therapeutic proteins. Very recently, it was reported a large protein conjugated to FA competes with the binding of FcRn with serum albumin, leading to limited serum half-life extension, because primary FA binding sites in serum albumin partially overlap with FcRn binding sites.

Intramolecular distance in the conjugate of urate oxidase and fatty acid governs FcRn binding and serum half-life in vivo.

Therapeutic proteins are indispensable for treatment of various human diseases. However, intrinsic short serum half-lives of proteins are still big hurdles for developing new therapeutic proteins or expanding applications of existing ones. Urate oxidase (Uox) is a therapeutic protein clinically used for treatment of hyperuricemia.

The structural basis of the low catalytic activities of the two minor β-carbonic anhydrases of the filamentous fungus Aspergillus fumigatus.

The β-carbonic anhydrases (β-CAs) are widely distributed zinc-metalloenzymes that play essential roles in growth, survival, development and virulence in fungi. The majority of filamentous ascomycetes possess multiple β-CA isoforms among which major and minor forms have been characterized. We examined the catalytic behavior of the two minor β-CAs, CafC and CafD, of Aspergillus fumigatus, and found that both enzymes exhibited low CO hydration activities.

Application of antihelix antibodies in protein structure determination.

Antibodies are indispensable tools in protein engineering and structural biology. Antibodies suitable for structural studies should recognize the 3-dimensional (3D) conformations of target proteins. Generating such antibodies and characterizing their complexes with antigens take a significant amount of time and effort.

Crystal Structure of a Highly Thermostable α-Carbonic Anhydrase from Persephonella marina EX-H1.

Bacterial α-type carbonic anhydrase (α-CA) is a zinc metalloenzyme that catalyzes the reversible and extremely rapid interconversion of carbon dioxide to bicarbonate. In this study, we report the first crystal structure of a hyperthermostable α-CA from Persephonella marina EXH1 ( CA) in the absence and presence of competitive inhibitor, acetazolamide. The structure reveals a compactly folded pm CA homodimer in which each monomer consists of a 10-stranded β-sheet in the center.

Ciclopirox inhibits Hepatitis B Virus secretion by blocking capsid assembly.

Chronic hepatitis B virus (HBV) infection can cause cirrhosis and hepatocellular carcinoma and is therefore a serious public health problem. Infected patients are currently treated with nucleoside/nucleotide analogs and interferon α, but this approach is not curative. Here, we screen 978 FDA-approved compounds for their ability to inhibit HBV replication in HBV-expressing HepG2.

Dynamic coordination of two-metal-ions orchestrates λ-exonuclease catalysis.

Metal ions at the active site of an enzyme act as cofactors, and their dynamic fluctuations can potentially influence enzyme activity. Here, we use λ-exonuclease as a model enzyme with two Mg binding sites and probe activity at various concentrations of magnesium by single-molecule-FRET. We find that while Mg and Mg have similar binding constants, the dissociation rate of Mg is two order of magnitude lower than that of Mg due to a kinetic-barrier-difference.

Crystal structure of the human glial fibrillary acidic protein 1B domain.

Glial fibrillary acidic protein (GFAP) is a homopolymeric type III intermediate filament (IF) that plays essential roles in cell migration, mitosis, development, and signaling in astrocytes and a specific type of glial cells. Its overexpression and genetic mutations lead to abnormal IF networks and accumulation of Rosenthal fibers, which results in the fatal neurodegenerative disorder Alexander disease. Herein, we present the first crystal structure of human GFAP spanning the central coiled-coil 1B domain at 2.

TAGLN2 polymerizes G-actin in a low ionic state but blocks Arp2/3-nucleated actin branching in physiological conditions.

TAGLN is an actin-binding protein family that comprises three isoforms with theorized roles in smooth muscle differentiation, tumour development, lymphocyte activation, and brain chemistry. However, their fundamental characteristics in regulation of the actin-based cytoskeleton are not fully understood. Here we show that TAGLN2 (including TAGLN1 and TAGLN3) extensively nucleates G-actin polymerization under low-salt conditions, where polymerization would be completely suppressed.

Structural insights into the oligomerization of FtsH periplasmic domain from Thermotoga maritima.

Prompt removal of misfolded membrane proteins and misassembled membrane protein complexes is essential for membrane homeostasis. However, the elimination of these toxic proteins from the hydrophobic membrane environment has high energetic barriers. The transmembrane protein, FtsH, is the only known ATP-dependent protease responsible for this task.