6 research outputs found
Clofarabine Targets the Large Subunit (α) of Human Ribonucleotide Reductase in Live Cells by Assembly into Persistent Hexamers
Available in PMC 2013 July 27Clofarabine (ClF) is a drug used in the treatment of leukemia. One of its primary targets is human ribonucleotide reductase (hRNR), a dual-subunit, (α2)m(β2)n, regulatory enzyme indispensable in de novo dNTP synthesis. We report that, in live mammalian cells, ClF targets hRNR by converting its α-subunit into kinetically stable hexamers. We established mammalian expression platforms that enabled isolation of functional α and characterization of its altered oligomeric associations in response to ClF treatment. Size exclusion chromatography and electron microscopy documented persistence of in-cell-assembled-α6. Our data validate hRNR as an important target of ClF, provide evidence that in vivo α's quaternary structure can be perturbed by a nonnatural ligand, and suggest small-molecule-promoted, persistent hexamerization as a strategy to modulate hRNR activity. These studies lay foundations for documentation of RNR oligomeric state within a cell.Howard Hughes Medical Institute (Investigator)National Institutes of Health (U.S.) (NIH grant GM29595)National Institutes of Health (U.S.) (NIH grant GM67167)Damon Runyon Cancer Research Foundation (postdoctoral fellowship (DRG2015-09))Howard Hughes Medical Institute (International Student Fellowship
3.3-Ă… resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound
Ribonucleotide reductases (RNRs) convert ribonucleotides into deoxyribonucleotides, a reaction essential for DNA replication and repair. Human RNR requires two subunits for activity, the α subunit contains the active site, and the β subunit houses the radical cofactor. Here, we present a 3.3-Å resolution structure by cryo-electron microscopy (EM) of a dATP-inhibited state of human RNR. This structure, which was determined in the presence of substrate CDP and allosteric regulators ATP and dATP, has three α 2 units arranged in an α 6 ring. At near-atomic resolution, these data provide insight into the molecular basis for CDP recognition by allosteric specificity effectors dATP/ATP. Additionally, we present lower-resolution EM structures of human α 6 in the presence of both the anticancer drug clofarabine triphosphate and β 2 . Together, these structures support a model for RNR inhibition in which β 2 is excluded from binding in a radical transfer competent position when α exists as a stable hexamer.National Institutes of Health (U.S.) (Grant GM29595
A soluble α-synuclein construct forms a dynamic tetramer
A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated “hairpin” structure of α-synuclein