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Structural basis for allosteric regulation of human ribonucleotide reductase by nucleotide-induced oligomerization

Abstract

Ribonucleotide reductase (RR) is an αnβn (RR1–RR2) complex that maintains balanced dNTP pools by reducing NDPs to dNDPs. RR1 is the catalytic subunit, and RR2 houses the free radical required for catalysis. RR is allosterically regulated by its activator ATP and its inhibitor dATP, which regulate RR activity by inducing oligomerization of RR1. Here, we report the first X-ray structures of human RR1 bound to TTP alone, dATP alone, TTP–GDP, TTP–ATP, and TTP–dATP. These structures provide insights into regulation of RR by ATP or dATP. At physiological dATP concentrations, RR1 forms inactive hexamers. We determined the first X-ray structure of the RR1–dATP hexamer and used single-particle electron microscopy to visualize the α6–ββ′–dATP holocomplex. Site-directed mutagenesis and functional assays confirm that hexamerization is a prerequisite for inhibition by dATP. Our data indicate a mechanism for regulating RR activity by dATP-induced oligomerization.

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Figure 1: Structure of hRRM1.
Figure 2: ATP and dATP binding at the A site of hRRM1.
Figure 3: SEC analysis of hRRM1 oligomers and enzyme activities of wild-type and mutant hRRM1.
Figure 4: Hexameric packing of RR1 based on the low-resolution X-ray crystal structure of the ScRR1 hexamer.
Figure 5: Electron microscopy of the α6–ββ′–dATP holocomplex.

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Acknowledgements

We thank members of the GMCA-CAT, NE-CAT and BIOCARS beamlines at the Advanced Photon Source at the Argonne National Laboratory and X29 at National Synchrotron Light Source at Brookhaven National Lab for assistance with data collection. We thank Y. Yen at the City of Hope Hospital, Los Angeles, for the gift of plasmid carrying the cDNA of hRRM1. We also thank C. Faber, M. Maguire, B. Cooperman and A. Hofer for useful discussion. This research was supported by US National Institutes of Health grants 2R01CA100827-04A1, 3R01CA100827-07S1 (to C.G.D.) and grants from the Swedish Research Council and Swedish Cancer Society (to P.N.). The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck, the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust. T.W. is funded by the Howard Hughes Medical Institute. We thank A. Berges at Wyatt Corporation for assisting us with MALS experiments and J. Stubbe and her group for teaching us the RR functional assay.

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J.W.F. purified protein, collected X-ray data and determined hRR1 and ScRR1 structures. S.R.W. purified protein, crystallized the hexamer, collected X-ray data, and did all the biochemical experiments. M.F.A. conducted the CD and fluorescence experiments. H.X. collected data and determined hexamer structure. R.N. and S.J. helped in protein purification. J.P. helped with SEC. R.M.W., S.F. and A.R. conducted crystallography and SEC studies of truncated hRR1. P.N. directed research on the truncated hRR1. Z.L. collected EM data and did image reconstruction. T.W. directed EM experiments. J.W.F., S.R.W., M.F.A., P.N. and T.W. helped with manuscript preparation. C.G.D. directed research, data analysis and manuscript preparation.

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Correspondence to Chris Godfrey Dealwis.

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Supplementary Discussion (including three figures and two tables), Supplementary Methods (including six figures and two tables), Supplementary Tables 1 and 2, and Supplementary Figures 1–5 (PDF 2590 kb)

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Fairman, J., Wijerathna, S., Ahmad, M. et al. Structural basis for allosteric regulation of human ribonucleotide reductase by nucleotide-induced oligomerization. Nat Struct Mol Biol 18, 316–322 (2011). https://doi.org/10.1038/nsmb.2007

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