TY - JOUR
T1 - Allosteric Inhibition of Human Ribonucleotide Reductase by dATP Entails the Stabilization of a Hexamer
AU - Ando, Nozomi
AU - Li, Haoran
AU - Brignole, Edward J.
AU - Thompson, Samuel
AU - McLaughlin, Martin I.
AU - Page, Julia E.
AU - Asturias, Francisco J.
AU - Stubbe, Jo Anne
AU - Drennan, Catherine L.
N1 - Funding Information:
This work was supported by the National Institutes of Health Grants GM100008 (N.A.) and GM29595 (J.S.) and MIT''s Undergraduate Research Opportunities Program (M.I.M., J.E.P., and S.T.). C.L.D. is a Howard Hughes Medical Institute Investigator. SAXS and crystallography data collection were conducted at the Cornell High Energy Synchrotron Source (CHESS), which is supported by the National Science Foundation Award DMR-1332208, using the Macromolecular Diffraction at CHESS (MacCHESS) facility, which is supported by award GM-103485 from the National Institute of General Medical Sciences, National Institutes of Health. We thank Chae Un Kim (CHESS/UNIST) for assistance with the collection of diffraction data that yielded crystal structure, Yimon Aye (Cornell) for purifying the sample that was used in crystallization and EM, and Marco Jost (MIT) and Bob Grant (MIT) for assistance with data processing and refinement. We are grateful to Richard Gillilan (CHESS) for setting up the SAXS flow cell and robotics, Cynthia Kinsland (Cornell) for providing advice on purification, and Michael Funk for assistance with the preparation of electron microscopy specimens.
Publisher Copyright:
© 2015 American Chemical Society.
PY - 2016/1/19
Y1 - 2016/1/19
N2 - 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.
AB - 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.
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U2 - 10.1021/acs.biochem.5b01207
DO - 10.1021/acs.biochem.5b01207
M3 - Article
C2 - 26727048
AN - SCOPUS:84955244638
SN - 0006-2960
VL - 55
SP - 373
EP - 381
JO - Biochemistry
JF - Biochemistry
IS - 2
ER -