title: Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer.
abstract: Ribonucleotide reductases (RNRs) are responsible for all de novo biosynthesis of DNA precursors in nature by catalyzing the conversion of ribonucleotides to deoxyribonucleotides. Because of its essential role in cell division, human RNR is a target for a number of anticancer drugs in clinical use. Like other class Ia RNRs, human RNR requires both a radical-generation subunit (β) and nucleotide-binding subunit (α) for activity. Because of their complex dependence on allosteric effectors, however, the active and inactive quaternary forms of many class Ia RNRs have remained in question. Here, we present an X-ray crystal structure of the human α subunit in the presence of inhibiting levels of dATP, depicting a ring-shaped hexamer (α6) where the active sites line the inner hole. Surprisingly, our small-angle X-ray scattering (SAXS) results indicate that human α forms a similar hexamer in the presence of ATP, an activating effector. In both cases, α6 is assembled from dimers (α2) without a previously proposed tetramer intermediate (α4). However, we show with SAXS and electron microscopy that at millimolar ATP, the ATP-induced α6 can further interconvert with higher-order filaments. Differences in the dATP- and ATP-induced α6 were further examined by SAXS in the presence of the β subunit and by activity assays as a function of ATP or dATP. Together, these results suggest that dATP-induced α6 is more stable than the ATP-induced α6 and that stabilization of this ring-shaped configuration provides a mechanism to prevent access of the β subunit to the active site of α.
authors.forename: Nozomi
authors.initials: N
authors.lastname: Ando
authors.forename: Haoran
authors.initials: H
authors.lastname: Li
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Brignole
authors.forename: Samuel
authors.initials: S
authors.lastname: Thompson
authors.forename: Martin I
authors.initials: MI
authors.lastname: McLaughlin
authors.forename: Julia E
authors.initials: JE
authors.lastname: Page
authors.affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute , La Jolla, California 92037, United States.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Asturias
authors.forename: JoAnne
authors.initials: J
authors.lastname: Stubbe
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Drennan
mesh_terms: D000494:Allosteric Regulation; D018360:Crystallography, X-Ray; D003838:Deoxyadenine Nucleotides / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D003838:Deoxyadenine Nucleotides; D012264:Ribonucleotide Reductases; C026600:2'-deoxyadenosine triphosphate