ATP-sensitive potassium channels (K) are energy sensors on the plasma membrane. By sensing the intracellular ADP/ATP ratio of β-cells, pancreatic K channels control insulin release and regulate metabolism at the whole body level. They are implicated in many metabolic disorders and diseases and are therefore important drug targets. Here, we present three structures of pancreatic K channels solved by cryo-electron microscopy (cryo-EM), at resolutions ranging from 4.1 to 4.5 Å. These structures depict the binding site of the antidiabetic drug glibenclamide, indicate how Kir6.2 (inward-rectifying potassium channel 6.2) N-terminus participates in the coupling between the peripheral SUR1 (sulfonylurea receptor 1) subunit and the central Kir6.2 channel, reveal the binding mode of activating nucleotides, and suggest the mechanism of how Mg-ADP binding on nucleotide binding domains (NBDs) drives a conformational change of the SUR1 subunit.
Adenosine Triphosphate ; metabolism ; Amino Acid Sequence ; Animals ; Binding Sites ; Cryoelectron Microscopy ; Ligands ; Mesocricetus ; Mice ; Models, Molecular ; Nucleotides ; metabolism ; Pancreas ; metabolism ; Potassium Channels, Inwardly Rectifying ; chemistry ; metabolism ; Protein Binding ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Subunits ; chemistry ; metabolism ; Sf9 Cells ; Spodoptera ; Sulfonylurea Receptors ; chemistry ; metabolism

Animals ; Cell Respiration ; Electron Transport Complex III ; metabolism ; Humans ; Iron-Sulfur Proteins ; chemistry ; metabolism ; Mitochondria ; metabolism ; Peptide Fragments ; chemistry ; metabolism ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Subunits ; chemistry

MRG proteins are conserved during evolution in fungi, flies, mammals and plants, and they can exhibit diversified functions. The animal MRGs were found to form various complexes to activate gene expression. Plant MRG1/2 and MRG702 were reported to be involved in the regulation of flowering time via binding to H3K36me3-marked flowering genes. Herein, we determined the crystal structure of MRG701 chromodomain (MRG701). MRG701 forms a novel dimerization fold both in crystal and in solution. Moreover, we found that the dimerization of MRG chromodomains is conserved in green plants. Our findings may provide new insights into the mechanism of MRGs in regulation of gene expression in green plants.
Amino Acid Sequence ; Arabidopsis ; genetics ; metabolism ; Arabidopsis Proteins ; chemistry ; genetics ; metabolism ; Binding Sites ; Chromosomal Proteins, Non-Histone ; chemistry ; genetics ; metabolism ; Cloning, Molecular ; Crystallography, X-Ray ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Histones ; chemistry ; genetics ; metabolism ; Models, Molecular ; Oryza ; genetics ; metabolism ; Peptides ; chemistry ; genetics ; metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Isoforms ; chemistry ; genetics ; metabolism ; Protein Multimerization ; Protein Structure, Secondary ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Sequence Homology, Amino Acid ; Viridiplantae ; genetics ; metabolism

Cell Cycle Proteins ; chemistry ; Histones ; chemistry ; Humans ; Minichromosome Maintenance Complex Component 2 ; chemistry ; Models, Molecular ; Protein Multimerization ; Protein Structure, Quaternary

Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobacterium tuberculosis (Mtb). Consequently, stalling dehydration cures tuberculosis (TB). Clinically used anti-TB drugs like thiacetazone (TAC) and isoxyl (ISO) as well as flavonoids inhibit the enzyme activity of the β-hydroxyacyl-ACP dehydratase HadAB complex. How this inhibition is exerted, has remained an enigma for years. Here, we describe the first crystal structures of the MtbHadAB complex bound with flavonoid inhibitor butein, 2',4,4'-trihydroxychalcone or fisetin. Despite sharing no sequence identity from Blast, HadA and HadB adopt a very similar hotdog fold. HadA forms a tight dimer with HadB in which the proteins are sitting side-by-side, but are oriented anti-parallel. While HadB contributes the catalytically critical His-Asp dyad, HadA binds the fatty acid substrate in a long channel. The atypical double hotdog fold with a single active site formed by MtbHadAB gives rise to a long, narrow cavity that vertically traverses the fatty acid binding channel. At the base of this cavity lies Cys61, which upon mutation to Ser confers drug-resistance in TB patients. We show that inhibitors bind in this cavity and protrude into the substrate binding channel. Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site. The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs.
Amino Acid Sequence ; Bacterial Proteins ; chemistry ; metabolism ; Catalytic Domain ; Enzyme Inhibitors ; chemistry ; pharmacology ; Flavonoids ; chemistry ; pharmacology ; Hydro-Lyases ; antagonists & inhibitors ; chemistry ; Molecular Sequence Data ; Mycobacterium tuberculosis ; drug effects ; enzymology ; Protein Binding ; drug effects ; Protein Multimerization ; drug effects ; Protein Structure, Secondary ; Sequence Alignment

Allosteric Regulation ; Carrier Proteins ; chemistry ; genetics ; metabolism ; Cell Proliferation ; Gene Expression ; Glycolysis ; genetics ; Humans ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Mutation ; Neoplasms ; enzymology ; genetics ; pathology ; Oxidative Phosphorylation ; Protein Conformation ; Protein Multimerization ; Protein Subunits ; chemistry ; genetics ; metabolism ; Thyroid Hormones ; chemistry ; genetics ; metabolism ; Tumor Cells, Cultured

Animals ; COS Cells ; Cercopithecus aethiops ; Endoplasmic Reticulum ; GTP Phosphohydrolases ; antagonists & inhibitors ; chemistry ; genetics ; metabolism ; GTP-Binding Proteins ; antagonists & inhibitors ; chemistry ; genetics ; metabolism ; Gene Expression ; Genetic Complementation Test ; HeLa Cells ; Humans ; Kinetics ; Membrane Fusion ; genetics ; Membrane Proteins ; antagonists & inhibitors ; chemistry ; genetics ; metabolism ; Protein Multimerization ; RNA, Small Interfering ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism ; Vesicular Transport Proteins ; genetics ; metabolism

Pyruvate kinase isoform M2 (PKM2) converts phosphoenolpyruvate (PEP) to pyruvate and plays an important role in cancer metabolism. Here, we show that post-translational modifications and a patient-derived mutation regulate pyruvate kinase activity of PKM2 through modulating the conformation of the PKM2 tetramer. We determined crystal structures of human PKM2 mutants and proposed a "seesaw" model to illustrate conformational changes between an inactive T-state and an active R-state tetramers of PKM2. Biochemical and structural analyses demonstrate that PKM2(Y105E) (phosphorylation mimic of Y105) decreases pyruvate kinase activity by inhibiting FBP (fructose 1,6-bisphosphate)-induced R-state formation, and PKM2(K305Q) (acetylation mimic of K305) abolishes the activity by hindering tetramer formation. K422R, a patient-derived mutation of PKM2, favors a stable, inactive T-state tetramer because of strong intermolecular interactions. Our study reveals the mechanism for dynamic regulation of PKM2 by post-translational modifications and a patient-derived mutation and provides a structural basis for further investigation of other modifications and mutations of PKM2 yet to be discovered.
Acetylation ; Allosteric Regulation ; Carrier Proteins ; chemistry ; genetics ; metabolism ; Crystallography, X-Ray ; Fructosediphosphates ; chemistry ; metabolism ; Gene Expression ; Humans ; Kinetics ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Models, Molecular ; Mutation ; Neoplasms ; enzymology ; genetics ; pathology ; Phosphorylation ; Protein Conformation ; Protein Multimerization ; Protein Processing, Post-Translational ; Protein Subunits ; chemistry ; genetics ; metabolism ; Thyroid Hormones ; chemistry ; genetics ; metabolism ; Tumor Cells, Cultured

Recombinant protein SMB(PRG4) containing two Somatomedin B domains and a small amount of glycosylation of repetitive sequences of proteoglycan 4 was cloned according to PGR4 gene polymorphism. Mature purification process was established and recombinant protein SMB(PRG4), with high-level expression was purified. By using size-exclusion chromatogaraphy and dynamic light scattering, we found that the recombinant protein self-aggregate to dimeric form. Structure prediction and non-reducing electrophoresis revealed that SMB(PRG4), was a non-covalently bonded dimer.
Glycosylation ; Protein Multimerization ; Proteoglycans ; chemistry ; Recombinant Proteins ; chemistry ; Somatomedins ; chemistry

Binding Sites ; Crystallography, X-Ray ; Humans ; Models, Molecular ; Protein Interaction Domains and Motifs ; Protein Multimerization ; Protein Structure, Secondary ; Receptors, Metabotropic Glutamate ; chemistry ; physiology