title: Glutamate 52-β at the α/β subunit interface ofEscherichia coliclass Ia ribonucleotide reductase is essential for conformational gating of radical transfer.
abstract: Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside diphosphate substrates (S) to deoxynucleotides with allosteric effectors (e) controlling their relative ratios and amounts, crucial for fidelity of DNA replication and repair.Escherichia coliclass Ia RNR is composed of α and β subunits that form a transient, active α2β2 complex. TheE. coliRNR is rate-limited by S/e-dependent conformational change(s) that trigger the radical initiation step through a pathway of 35 Å across the subunit (α/β) interface. The weak subunit affinity and complex nucleotide-dependent quaternary structures have precluded a molecular understanding of the kinetic gating mechanism(s) of the RNR machinery. Using a docking model of α2β2 created from X-ray structures of α and β and conserved residues from a new subclassification of theE. coliIa RNR (Iag), we identified and investigated four residues at the α/β interface (Glu350and Glu52in β2 and Arg329and Arg639in α2) of potential interest in kinetic gating. Mutation of each residue resulted in loss of activity and with the exception of E52Q-β2, weakened subunit affinity. An RNR mutant with 2,3,5-trifluorotyrosine radical (F3Y122•) replacing the stable Tyr122•in WT-β2, a mutation that partly overcomes conformational gating, was placed in the E52Q background. Incubation of this double mutant with His6-α2/S/e resulted in an RNR capable of catalyzing pathway-radical formation (Tyr356•-β2), 0.5 eq of dCDP/F3Y122•, and formation of an α2β2 complex that is isolable in pulldown assays over 2 h. Negative stain EM images with S/e (GDP/TTP) revealed the uniformity of the α2β2 complex formed.
authors.affiliation: From the Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou 310058, China and.
authors.forename: Qinghui
authors.initials: Q
authors.lastname: Lin
authors.affiliation: the Departments of Chemistry and.
authors.forename: Mackenzie J
authors.initials: MJ
authors.lastname: Parker
authors.affiliation: the Departments of Chemistry and.
authors.forename: Alexander T
authors.initials: AT
authors.lastname: Taguchi
authors.affiliation: the Departments of Chemistry and.
authors.forename: Kanchana
authors.initials: K
authors.lastname: Ravichandran
authors.affiliation: the Departments of Chemistry and.
authors.forename: Albert
authors.initials: A
authors.lastname: Kim
authors.affiliation: the Departments of Chemistry and.
authors.forename: Gyunghoon
authors.initials: G
authors.lastname: Kang
authors.affiliation: From the Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou 310058, China and.
authors.forename: Jimin
authors.initials: J
authors.lastname: Shao
authors.affiliation: the Departments of Chemistry and cdrennan@mit.edu.|Biology, and.|Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139.
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Drennan
authors.affiliation: the Departments of Chemistry and stubbe@mit.edu.|Biology, and.
authors.forename: JoAnne
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D019943:Amino Acid Substitution; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D062105:Molecular Docking Simulation; D020125:Mutation, Missense; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; C571544:NrdB protein, E coli; D012264:Ribonucleotide Reductases
keywords: conformational change; electron microscopy (EM); electron paramagnetic resonance (EPR); enzyme mutation; glutamate; radical transfer; ribonucleotide reductase; subunit interface